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Fish protein hydrolysates: Proximate composition, amino acid composition,

antioxidant activities and applications: A review

Article  in  Food Chemistry · December 2012

DOI: 10.1016/j.foodchem.2012.06.100 · Source: PubMed

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 1 Fish protein hydrolysates: proximate composition, amino acid
2 composition, antioxidant activities and applications
3
4
5 M. Chalamaiaha, B. Dinesh kumara, R. Hemalathab and T. Jyothirmayic,*
6
a
7 Food and Drug Toxicology Research Centre, National Institute of Nutrition (Indian
8 Council of Medical Research), Jamai-osmania, Hyderabad – 500 604, India
9
b
10 Microbiology and Immunology Division, National Institute of Nutrition (Indian Council
11 of Medical Research), Jamai-osmania, Hyderabad – 500 604, India
12
c
13 Central Food Technological Research Institute (Council of Scientific and Industrial
14 Research), Resource Centre, Hubshiguda, Uppal Road, Hyderabad-500 007, India
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19
20
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23 ACCEPTED MANUSCRIPT – FOOD CHEMISTRY
24
25
26 Please cite as:
27 M. Chalamaiah, B. Dinesh Kumar, R. Hemalatha and T.
28 Jyothirmayi. (2012). Fish protein hydrolysates: proximate
29 composition, amino acid composition, antioxidant activities
30 and applications: a review. Food Chemistry, 135, 3020-3038.
31
32
33
34 E-mail address:
35 Chalamaiah.m@gmail.com (M. Chalamaiah) – Paper with full

36 bibliography can be requested at this e.mail id.

37
38
39

1
40
41 ABSTRACT
42
43 Fish processing industry produces more than 60% by-products as waste, which includes

44 skin, head, viscera, trimmings, liver, frames, bones, and roes. These by-product wastes

45 contain good amount of protein rich material that are normally processed into low

46 market-value products, such as animal feed, fish meal and fertilizer. In view of utilizing

47 these fish industry wastes, and for increasing the value to several underutilized fish

48 species, protein hydrolysates from fish proteins are being prepared by several researchers

49 all over the world. Fish protein hydrolysates are breakdown products of enzymatic

50 conversion of fish proteins into smaller peptides, which normally contain 2 to 20 amino

51 acids. In recent years, fish protein hydrolysates have attracted much attention of food

52 biotechnologists due to the availability of large quantities of raw material for the process,

53 and presence of high protein content with good amino acid balance and bioactive

54 peptides (antioxidant, antihypertensive, immunomodulatory and antimicrobial peptides).

55 _______________________________________________________________________
56 Keywords:
57 Fish
58 Fish by-product waste
59 Fish protein hydrolysates
60 Chemical composition
61 Amino acid composition
62 Antioxidant peptides
63 Fish protein hydrolysates applications
64
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67
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69
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71
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73

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74
75 1. Introduction
76
77 Fish industry is a major economic source for a number of countries worldwide.

78 Fish protein is an essential source of nutrients for many people, especially in developing

79 countries. It is estimated that worldwide, one billion people depend on producing,

80 processing and trading fish for their livelihood (Oosterveer, 2008). Fish processing

81 industry produces more than 60% by-products as waste, which includes head, skin,

82 trimmings, fins, frames, viscera and roes, and only 40% fish products for human

83 consumption (Dekkers, Raghavan, Kristinsson, & Marshall, 2011). These large quantities

84 of fish by-product waste from fisheries would create serious pollution and disposal

85 problems in both developed and developing countries. These by-product wastes contain

86 good amount of protein rich material that are normally processed into low market-value

87 products, such as animal feed, fish meal and fertilizer (Hsu, 2010). In view of utilizing

88 these protein rich fish processing by-product wastes, several biotechniques have been

89 developed to recover the essential nutrients and bioactive compounds that would help the

90 improvement of human health by protecting from several diseases and providing essential

91 nutrients for maintaining good health, and to solve the pollution and disposal problems as

92 well. The biotechniques which are currently employed to recover the nutritional and

93 physiological important peptides are enzymatic hydrolysis of fish proteins that results in

94 the production of biologically active protein hydrolysates from these commercially low

95 value fish processing by-products and underutilized fish species. Several proteolytic

96 enzymes are most commonly used to hydrolyze the fish proteins for the production of

97 fish protein hydrolysates. These include Alcalase, papain, pepsin, trypsin, α -

98 chymotrypsin, pancreatin, Flavourzyme, Pronase, Neutrase, Protamex, bromelain, cryotin

3
 99 F, protease N, protease A, Orientase, thermolysin, and Validase (Je, Lee, Lee, & Ahn,

100 2009; Ren et al., 2008; Ngo, Qian, Ryu, Park, & Kim, 2010; Hsu, 2010; Samaranayaka &

101 Li-chan, 2008; Raghavan & Kristinsson, 2008). With the advent of these enzymatic

102 techniques, several fish protein hydrolysates are produced from various protein rich fish

103 by-product wastes (Pastoriza, Sampedro, Cabo, Herrera, & Bernardez, 2004; Dong,

104 Sheng, Fu, & Wen, 2005; Wasswa, Tang, Gu, & Yuan, 2007; Beaulieu, Thibodeau, Bryl,

105 & Carbonneau, 2009; Duan, Wang, Yi, & Yin, 2010; Ahn, Lee, & Je, 2010) and have a

106 variety of applications including pharmaceutical products, cosmetics, and animal

107 nutrition (Wergedahl, Liaset, Gudbrandsen, Lied, Espe, & Muna, 2004; Oliva-Teles,

108 Cerqueira, & Gonçalves, 1999).

109 Protein hydrolysates are breakdown products of enzymatic conversion of proteins

110 into smaller peptides. Generally protein hydrolysates are small fragments of peptides that

111 contain 2 to 20 amino acids. These protein hydrolysates are produced by the enzymatic

112 hydrolysis of native proteins. Protein hydrolysis decreases the peptide size, and thereby

113 making hydrolysates the most available amino acid source for various physiological

114 functions of human body. Protein hydrolysates are used as readily available sources of

115 protein for humans and animals due to their good functional properties (Neklyudov,

116 Ivankin, & Berdutina, 2000).

117 Proper utilization of protein rich fish processing wastes and underutilized fish

118 species could be achieved by converting these materials into fish protein hydrolysates.

119 The conversion of inexpensive fish processing by-products and underutilized fish

120 proteins into valuable products is of great interest in recent times to food scientists all

121 over the world. Currently, fish protein hydrolysates are considered the most important

4
122 source of protein and bioactive peptides. The production and functional properties of fish

123 protein hydrolysates have been reviewed by Kristinsson and Rasco (2000b). The use and

124 application of protein hydrolysates in human nutrition have been reported in literature

125 (Neklyudov et al., 2000; Clemente, 2000). Many studies reported the biological activity

126 of various fish protein hydrolysates in vivo and in vitro by means of their bioactive

127 peptides (Je, Park, Kwon, & Kim, 2004; Duartea, Vinderola, Ritzc, Perdigon, & Matara,

128 2006; Picot et al., 2006; Liaset et al., 2009).

129 In recent years, several attempts have been made for utilization of the protein rich

130 fish processing by-product wastes and underutilized fish proteins for the production of

131 commercially valuable food ingredients. Fish protein hydrolysates with good nutritional

132 composition, amino acid profile, and antioxidant activities have gained great attention of

133 food scientists. Due to the presence of essential nutrients and bioactive components in

134 fish protein hydrolysates, these find place in various industrial applications. This paper

135 reviews the recent research on fish protein hydrolysates and their proximate composition,

136 amino acid composition, and antioxidant activities. Additionally, the potential

137 applications of fish protein hydrolysates are presented.

138
139
140 1.1. Fish skin protein hydrolysates
141
142 Fish skin, a processing by-product waste from fish industry, is a rich source of

143 collagen and gelatin. There are several studies which demonstrate the potential of fish

144 skin processing by-product waste to convert into fish protein hydrolysates. A number of

145 studies reported the preparation of protein hydrolysates from fish skin waste of different

146 species. These include Grass carp (Ctenopharyngodon idella) (Wasswa et al., 2007),

5
147 Rachycentron canadum (Yang, Ho, Chu, & Chow, 2008), Tilapia (Yang, Liang, Chow, &

148 Siebert, 2009), sole (Gimenez, Aleman, Montero, & Gomez-Guillen, 2009), Lutjanus

149 vitta (Khantaphant & Benjakul, 2008), Theragra chalcogramma (Zhuang, Li, & Zhao,

150 2009), Priacanthus macracanthus (Phanturat, Benjakul, Visessanguan, & Roytrakul,

151 2010), North Atlantic lean fish (Picot et al., 2010), Channel catfish (Ictalurus punctatus)

152 (Yin, Pu, Wan, Xiang, Bechtel, & Sathivel, 2010), Oreochromis niloticus (Ngo et al.,

153 2010), Aphanopus carbo (Batista, Ramos, Coutinho, Bandarra, & Nunes, 2010), Raja

154 kenojei (Lee, Jeon, & Byun, 2011), Magalaspis cordyla and Otolithes ruber (Sampath

155 Kumar, Nazeer, & Jaiganesh, 2011).

156 Wasswa, Tang, and Gu (2008) and Wasswa et al. (2007) prepared protein

157 hydrolysates from grass carp skin using Alcalase and optimized the hydrolysis conditions

158 by using a response surface methodology (RSM). Jian-xin and Zheng (2009) investigated

159 the enzymatic hydrolysis of Gadus morrhua skin collagen and studied the molecular

160 weight distribution of hydrolysates. Yin et al. (2010) have reported cat fish skin isolate

161 soluble and insoluble protein hydrolysates from hydrolyzed catfish skin and described the

162 rheological and functional properties of the protein hydrolysates. Another study reported

163 by Aleman, Gimenez, Montero, and Gomez-Guillen (2011) attempted to prepare the

164 protein hydrolysates from Tuna and Halibut skin gelatins. Recently, Sampath Kumar et

165 al. (2011) reported the protein hydrolysates from skin of two marine fishes, horse

166 mackerel (Magalaspis cordyla) and croaker (Otolithes ruber), by using gastrointestinal

167 proteases (pepsin, trypsin and α-chymotrpsin).

168

169

6
170 1.2. Fish head protein hydrolysates

171 Fish processing industries generate a considerable amount of protein rich fish

172 heads as a by-product waste. Protein hydrolysates from fish head by-product waste have

173 been prepared from various species of fishes; these include salmon (Salmo salar)

174 (Gbogouri, Linder, Fanni, & Parmentier, 2004), Red salmon (Oncorhynchus nerka)

175 (Sathivel, Smiley, Prinyawiwatkul, & Bechtel, 2005), Sardinella aurita (Souissi,

176 Bougatef, Triki-Ellouz, & Nasri, 2007), Aphanopus carbo (Batista et al., 2010),

177 Sardinella aurita (Bougatef et al., 2010).

178 Sathivel et al. (2003) demonstrated the Herring (Clupea harengus) protein

179 hydrolysates from head, whole fish, body, and gonad after 75 min digestion using

180 Alcalase as proteolytic enzyme and reported the functional properties of hydrolysates.

181 Gbogouri et al. (2004) obtained protein hydrolysates from salmon heads by enzymatic

182 treatment with Alcalase and optimized temperature, enzyme to substrate ratio, and pH

183 leading to various hydrolysates (11.5% to 17.3% degree of hydrolysis) using response

184 surface methodology (RSM). A study conducted by Sathivel et al. (2005) reported the

185 protein hydrolysates from red salmon (Oncorhynchus nerka) head and studied the effects

186 of different proteolytic enzymes (Alcalase, Flavourzyme, palatase, and Neutrase) and

187 different reaction durations (25, 50, 75 min) on functional and nutritional properties of

188 red salmon (Oncorhynchus nerka) head hydrolysates. Bougatef et al. (2008) obtained

189 protein hydrolysates from heads and viscera of sardinella (Sardinella aurita) by treatment

190 with Alcalase, chymotrypsin, crude enzyme preparations from Bacillus licheniformis

191 NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina

192 pilchardus) viscera. Another study carried out by Souissi et al. (2007) have described fish

7
193 protein hydrolysates with different degrees of hydrolysis (DH of 6.62, 9.31 and 10.16)

194 prepared from heads and viscera of sardinella (Sardinella aurita) by treatment with

195 Alcalase.

196
197 1.3. Fish muscle protein hydrolysates

198 Protein rich dark muscles from fishes have limited uses due to their accessibility

199 to oxidation and off-flavour. They are mostly processed into low market-value products.

200 Therefore, conversion of these protein rich materials into protein hydrolysates for further

201 utilization may produce highly valuable products (Hsu, 2010). Several protein

202 hydrolysates are produced using various fish muscle proteins from several fish species

203 such as Atlantic Salmon (Salmo salar) (Kristinsson & Rasco, 2000a), Merluccius

204 productus (Pacheco-Aguilar, Mazorra-Manzano, & Ramirez-Suarez, 2008), Decapters

205 maruadsi (Thiansilakul, Benjakul, & Shahidi, 2007), Selaroides leptolepis (Klompong,

206 Benjakul, Kantachote, & Shahidi, 2007), Ctenopharyngodon idellus (Ren et al., 2008),

207 Hypophthalmichthys molitrix (Dong, Zeng, Wang, Liu, Zhao, & Yang, 2008),

208 Oreochromis niloticus (Raghavan & Kristinsson, 2008), Mustelus mustelus (Bougatef,

209 Hajji, Balti, Lassoued, Triki-Ellouz, & Nasri, 2009), Pollachius virens (Chabeaud,

210 Dutournie, Guerard, Vandanjon, & Bourseau, 2009), Salmo salar; Onchorhynchus

211 kisutch; Theragra chalcogramma; Micromesistius australis (Nakajima, Yoshie-Stark, &

212 Ogushi, 2009), Thunnus tonggol (Hsu, 2010), Lepturacanthus savala and Sphyraena

213 barracuda (Rajaram & Nazeer, 2010), Nemipterus hexodon (Nalinanon, Benjakul,

214 Kishimura, & Shahidi, 2011), Nemipterus japonicus (Naqash & Nazeer, 2011a) and

215 Channa striatus (Ghassem, Fern, Said, Ali, Ibrahim, & Babji, 2011).

8
216 Spray-dried protein hydrolysate from black tilapia (Oreochromis mossambicus)

217 flesh was prepared and evaluated the nutritional quality of protein hydrolysates (Abdul-

218 hamid, Bakar, & Bee, 2002). Another study carried out by Wu, Chen, and Shiau (2003)

219 described protein hydrolysates from Mackerel (Scomber austriasicus) meat by an

220 autolytic process and accelerated hydrolysis with a commercial enzyme, protease N and

221 investigated the changes in the levels and compositions of free amino acids and small

222 peptides during hydrolysis. Candido and Sgarbieri (2003) enzymatically hydrolyzed the

223 Nile tilapia (Oreochromus niloticus) myofibrillar proteins into protein hydrolysates with

224 different degree of hydrolysis (2.5, 3.5, 7.0, 9.0, 14.0 and 25.0%) using Flavourzyme.

225 Normah, Jamilah, Saari, and Yaakob (2005) produced protein hydrolysates from

226 threadfin bream (Nemipterus japonicus) by optimizing the hydrolysis conditions (pH,

227 temperature and enzyme [Alcalase] to substrate ratio). Martone, Borla, and Sanchez

228 (2005) prepared highly soluble fish protein hydrolysates with 80% protein from hake

229 (Merluccius hubssi) filleting waste. In a study performed by Theodore and Kristinsson

230 (2007) catfish muscle protein isolate was hydrolyzed and reported 5, 15 and 30% degrees

231 of hydrolysis using Protamex. Cinq-Mars and Li-Chan (2007) described the Pacific Hake

232 (Merluccius productus) fillet hydrolysate using Protamex protease under various

233 conditions of pH, hydrolysis time, and enzyme-to-substrate ratio (% E/S) according to a

234 response surface methodology study. Another study conducted by Pacheco-Aguilar et al.

235 (2008) used Pacific whiting (Merluccius productus) muscle to produce hydrolysates with

236 10, 15 and 20% degree of hydrolysis (DH) using the commercial protease Alcalase and

237 the protein hydrolysates were characterized for their solubility, emulsifying and foaming

238 properties at pH 4.0, 7.0 and 10. Cudennec, Ravallec-Ple, Courois, and Fouchereau-Peron

9
239 (2008) have described the protein hydrolysates from blue whiting (Merliccius poutassou)

240 muscle. Klompong, Benjakul, Yachai, Visessanguan, Shahidi, and Hayes (2009a)

241 obtained protein hydrolysates from yellow stripe trevally (Selaroides leptolepis) meat

242 with 15% degree of hydrolysis (DH) using Alcalase and Flavourzyme following pH-stat

243 method. Choi, Hur, Choi, Konno, and Park (2009) have prepared protein hydrolysates

244 from protein isolate of frozen small croaker (Pennahia argentata) muscle using Alcalase,

245 Neutrase, Protamex, and Flavourzyme. Protein hydrolysates from meat of Misgurnus

246 anguillicaudatus have been reported (You, Zhao, Cui, Zhao, & Yang, 2009; You, Zhao,

247 Regenstein, & Ren, 2010a; You, Zhao, Regenstein, & Ren, 2010b). A study conducted by

248 Raghavan and Kristinsson (2009) solubilized the Tilapia fillet protein waste and

249 hydrolysed using two enzymes, cryotin-F or Flavourzyme, to 7.5% and 25% degree of

250 hydrolysis (DH) and produced Tilapia protein hydrolysates. A study conducted by Foh,

251 Kamara, Amadou, Foh, and Wenshui (2011) reported protein hydrolysates from minced

252 meat of Tilapia (Oreochromis niloticus). Geirsdottir et al. (2011) investigated the

253 enzymatic hydrolysis of blue whiting (Micromesistius poutassou) proteins with Alcalase.

254 Santos, Martins, Salas-Mellado, and Prentice (2011) have described the protein

255 hydrolysates from bluewing searobin (Prionotus punctatus) fillets using Alcalase and

256 Flavourzyme. Recently, Khantaphant, Benjakul, and Kishimura (2011) reported protein

257 hydrolysates from the muscle of brownstripe red snapper (Lutjanus vitta) prepared by

258 using Alcalase or Flavourzyme as the first step with 40% degree of hydrolysis (DH),

259 followed by hydrolysis with pyloric caeca protease (isolated from Lutjanus vitta) as the

260 second step. Another study carried out by Nalinanon et al. (2011) investigated protein

261 hydrolysates prepared from ornate threadfin bream (Nemipterus hexodon) muscle, using

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262 skipjack tuna pepsin with different degrees of hydrolysis (DH: 10, 20 and 30%). A study

263 conducted by Ghassem et al. (2011) hydrolyzed the sarcoplasmic and myofibrillar

264 proteins of Channa striatus muscle using proteinase K and thermolysin, and

265 characterized kinetic parameters for sarcoplasmic and myofibrillar protein hydrolysates.

266

267 1.4. Fish visceral protein hydrolysates

268
269 Fish viscera generated during processing is a potential source of protein that can

270 be used as a raw material for the production of protein hydrolysates, which may have

271 some exceptional properties for various industrial applications. In recent times, a number

272 of attempts have been made for the production of protein hydrolysates from fish viscera

273 using several proteolytic enzymes (Souissi et al., 2007; Bhaskar, Benila, Radha, & Lalitha,

274 2008; Ovissipour, Abedian Kenari, Motamedzadegan, Rasco, Safari, & Shahiri, 2009;

275 Bougatef et al., 2010; Batista et al., 2010; Hathwar et al., 2011).

276 Aspmo, Horn, and Eijsink (2005) studied the solubilization of cod (Gadus

277 morhua) visceral proteins at natural substrate pH using endogenous enzymes alone or in

278 combination with one of seven different commercial protease preparations (Alcalase,

279 Neutrase, Protamex, papain, bromelain, actinidin and a plant protease mix). Liceaga-

280 Gesualdo and Li-Chan (1999) described the protein hydrolysate from herring (Clupea

281 harengus) using an endopeptidase preparation from Bacillus licheniformis and reported

282 the degree of hydrolysis and functional properties of protein hydrolysates. Protein

283 hydrolysates have been produced from yellowfin tunas (Thunnus albacares) stomach by

284 a commercial neutral protease umamizyme (Guerard, Guimas, & Binet, 2002). Bhaskar

285 and Mahendrakar (2008) demonstrated the protein hydrolysates from visceral waste

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286 proteins of an Indian freshwater major carp catla (Catla catla) and optimized the

287 hydrolysis conditions (viz., time, temperature, pH and enzyme to substrate ratio) for

288 preparing protein hydrolysates from the fish visceral waste proteins. The visceral protein

289 of Persian sturgeon (Acipenser persicus) have been attempted to produce the protein

290 hydrolysates using Alcalase, Protamex, Neutrase, Flavourzyme, and trypsin enzymes and

291 reported the significant effect of hydrolysis conditions on protein hydrolysate properties

292 (Ovissipour et al., 2009). Another study performed by Ovissipour, Abedian Kenari,

293 Motamedzadegan, and Nazari (2010) reported the production of protein hydrolysates

294 from the viscera of yellowfin tuna (Thunnus albacares). Recently, Jai Ganesh, Nazeer,

295 and Sampath kumar (2011) have reported the hydrolysis of Parastromateus niger viscera

296 waste proteins into protein hydrolysates using gastrointestinal proteases such as pepsin,

297 trypsin and α-Chymotrpsin.

298
299 1.5. Fish liver protein hydrolysates

300 The increasing demand for utilization of limited natural resources and increasing

301 environmental pollution has emphasized the need for better utilization of fish processing

302 by-products. To maximize economical benefits, the efficient recovery and utilization of

303 by-products is very important. Je et al. (2009) reported protein hydrolysates from

304 skipjack tuna (Katsuwonus pelamis) liver, a fish by-product which is used to produce fish

305 meal and animal feed or is directly discarded as processing waste, by enzymatic

306 hydrolysis using commercial proteases such as Flavourzyme, Alcalase, Protamex, and

307 Neutrase. Another study performed by Ahn et al. (2010) has reported protein

308 hydrolysates from Tuna liver by enzymatic hydrolysis using various proteases such as

309 Alcalase, Neutrase and Protamex. The protein hydrolysates can be used as value-added

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310 products, which are widely applied to improve and upgrade the functional and nutritional

311 properties of proteins.

312
313 1.6. Fish bone protein hydrolysates

314 Fish backbone is one of the important parts of fish processing waste, and it

315 contains around 30% protein (Je, Qian, Byun, & Kim, 2007). Therefore, few researchers

316 attempted to prepare protein hydrolysates from underutilized fish backbone proteins in

317 order to make value added products. Sumaya-martinez, Castillo-morales, Favela-Torres,

318 Huerta-Ochoa, and Prado-Barragan (2005) studied the hydrolysis parameters for the

319 production of fish protein hydrolysates from gold carp (Carassius auratus) filleting by-

320 product (bone and muscle) using response surface methodology. A study conducted by Je

321 et al. (2007) reported the protein hydrolysates from tuna backbone protein using different

322 proteases such as Alcalase, α-chymotrypsin, Neutrase, papain, pepsin and trypsin.

323 Slizyte, Mozuraityte, Martinez-Alvarez, Falch, Fouchereau-Peron, and Rustad (2009)

324 conducted a series of hydrolysis trials using backbones from cod (Gadus morhua) that

325 were initially fresh or frozen and further hydrolysed for different times (10, 25, 45 and 60

326 min) and evaluated how storage and preparation of cod backbones influence the yield,

327 functionality, bioactivity and antioxidative properties of protein hydrolysates. Recently,

328 Nazeer, Deeptha, Jaiganesh, Sampathkumar, and Naqash (2011) hydrolyzed the

329 backbones of two commercially important fishes; seela (Sphyraena barracuda) and

330 ribbon fish (Lepturacanthus savala) using papain, pepsin and trypsin, and reported the

331 preparation of protein hydrolysates. Naqash and Nazeer (2011b) reported the protein

332 hydrolysates from Exocoetus volitans backbone prepared by using pepsin, papain and

333 trypsin.

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334 1.7. Fish frame protein hydrolysates

335 Several fish frame proteins are normally discarded as an industrial by-products in

336 the fish processing plants. Recently, various attempts have been made to produce protein

337 hydrolysates by several researchers to extract the commercially valuable products from

338 various fish frame by-products (Je, Park, & Kim, 2005; Jung et al., 2006; Lee, Qian, &

339 Kim, 2010).

340 Liaset, Lied, and Espe (2000) described the proteolysis of fish frames without

341 heads from Atlantic cod (Gadus morhua) and Atlantic salmon (Salmo salar) with the

342 industrial enzymes Neutrase, Alcalase and pepsin for 1, 15, 30, 45, 60, 90 and 120 min.

343 Another study carried out by Liaset, Julshamn, and Espe (2003) have hydrolyzed the

344 salmon frames without heads with the commercial protease mixture Protamex and

345 produced aqueous fraction rich in protein hydrolysates. Je et al. (2005) treated the Alaska

346 pollack frame protein with mackerel intestine crude enzyme and obtained Alaska pollack

347 frame protein hydrolysate. Rajapakse, Jung, Mendis, Moon, and Kim (2005) subjected

348 the yellowfin sole (Limanda aspera) frame containing considerable amount of protein

349 after filleting process to hydrolysis separately with seven different proteases, Alcalase,

350 Neutrase, pepsin, papain, α-chymotrypsin, trypsin and previously extracted tuna pyloric

351 caeca crude enzyme and obtained protein hydrolysates. Lee et al. (2010) have also

352 hydrolyzed the tuna frame protein using Alcalase, Neutrase, pepsin, papain, α-

353 chymotrypsin and trypsin and reported the protein hydrolysates. Recently, Hou, Li, Zhao,

354 Zhang, and Li (2011b) obtained protein hydrolysates from Alaska pollock frame by

355 hydrolyzing with ten different commercial proteases (Alcalase, Flavourzyme, acid

14
356 protease, Protamex, alkaline protease, trypsin, MEAP, neutral protease, bromelain and

357 papain).

358
359 1.8. Fish roe or egg protein hydrolysates

360 Roes or eggs of several fish species are underutilized and considered as by-

361 product waste in different parts of world, especially in developing countries. Not much

362 work was reported on utilization of fish roe except few value added products were

363 prepared (Bledsoe, Bledsoe, & Rasco, 2003; Chalamaiah, Balaswamy, Narsing Rao,

364 Prabhakara Rao, & Jyothirmayi, 2011). Fish roe contains considerable amount of protein,

365 in order to utilize this underutilized protein source from fish roe, protein hydrolysates

366 were prepared from underutilized fish (Cirrhinus mrigala) roe using Alcalase and papain

367 as proteolytic enzymes (Chalamaiah, Narsing Rao, Rao, & Jyothirmayi, 2010).

368

369 2. Proximate composition of fish protein hydrolysates

370
371 Chemical composition of food materials has an important role on human health in

372 supply of essential nutrients for maintaining prosperous health. Chemical composition of

373 fish protein hydrolysates is important in nutrition perspective of human health. Table 1

374 shows the proximate composition of fish protein hydrolysates. Several research

375 documents have been reported the chemical composition of fish protein hydrolysates that

376 are prepared from various fish protein sources from herring (Clupea harengus) (Hoyle &

377 Merritt, 1994), capelin (Mallotus villosus) (Shahidi, Han, & Synowiecki, 1995), pacific

378 whiting solid waste (Merluccius productus) (Benjakul & Morrissey, 1997), herring

379 (Clupea harengus) (Liceaga-Gesualdo & Li-Chan, 1999), atlantic salmon muscle (Salmo

380 salar) (Kristinsson & Rasco, 2000a), icelandic scallop (Chlamys islandica) (Mukhin,

15
381 Novikov, & Ryzhikova, 2001), black tilapia (Oreochromis mossambicus) (Abdul-Hamid

382 et al., 2002), herring byproduct (Clupea harengus) (Sathivel et al., 2003), salmon head

383 (Salmo salar) (Gbogouri et al., 2004), tuna byproduct (Nilsang, Lertsiri, Suphantharika,

384 & Assavanig, 2005), red salmon head (Oncorhynchus nerka) (Sathivel et al., 2005),

385 Saurida elongate (Dong et al., 2005), grass carp skin (Ctenopharyngodon idella)

386 (Wasswa et al., 2007), round scad (Decapterus maruadsi) (Thiansilakul et al., 2007),

387 sardinella by-product (Sardinella aurita) (Souissi et al., 2007), catla visceral waste

388 (Catla catla) (Bhaskar et al., 2008), pacific whiting muscle (Merluccius productus)

389 (Pacheco-Aguilar et al., 2008; Mazorra-Manzano, Pacheco-Aguilar, Ramirez-Suarez,

390 Garcia-Sanchez, & Lugo-Sanchez, 2011), croaker (Pennahia argentata) (Choi et al.,

391 2009), persian sturgeon viscera (Acipenser persicus) (Ovissipour et al., 2009), and

392 channel catfish skin (Ictalurus punctatus) (Yin et al., 2010), and Lutjanus vitta

393 (Khantaphant et al., 2011).

394 Many researchers reported the protein content of fish protein hydrolysates ranged

395 between 60 to 90 % of total composition (Sathivel et al., 2003; Nilsang et al., 2005; Dong

396 et al., 2005; Thiansilakul et al., 2007; Souissi et al., 2007; Pacheco-Aguilar et al., 2008;

397 Choi et al., 2009; Ovissipour et al., 2009; Khantaphant et al., 2011). The high protein

398 content reported for fish protein hydrolysates is due to solubilisation of proteins during

399 hydrolysis and removal of insoluble solid matter by centrifugation (Liceaga-Gesualdo &

400 Li-Chan, 1999; Chalamaiah et al., 2010). High protein content of fish protein

401 hydrolysates demonstrates its potential use as protein supplements for human nutrition.

402 Several studies reported the fat content for various fish protein hydrolysates were

403 below 5% (Hoyle & Merritt, 1994; Shahidi et al., 1995; Benjakul & Morrissey, 1997;

16
404 Liceaga-Gesualdo & Li-Chan, 1999; Kristinsson & Rasco, 2000a; Mukhin et al., 2001;

405 Abdul-Hamid et al., 2002; Sathivel et al., 2003; Gbogouri et al., 2004; Dong et al., 2005;

406 Nilsang et al., 2005; Wasswa et al., 2007; Thiansilakul et al., 2007; Pacheco-Aguilar et

407 al., 2008; Bhaskar et al., 2008; Ovissipour et al., 2009). Only few reports described the

408 fat content above 5% level for fish protein hydrolysates (Sathivel et al., 2005; Souissi et

409 al., 2007; Chalamaiah et al., 2010). The low fat content of fish protein hydrolysates is

410 because of removal of lipids with insoluble protein fractions by centrifugation.

411 Most of the studies demonstrated that protein hydrolysates from various fish

412 proteins contain moisture below 10% (Kristinsson & Rasco, 2000a; Mukhin et al., 2001;

413 Abdul-Hamid et al., 2002; Sathivel et al., 2003; Gbogouri et al., 2004; Dong et al., 2005;

414 Nilsang et al., 2005; Wasswa et al., 2007; Thiansilakul et al., 2007; Pacheco-Aguilar et

415 al., 2008; Bhaskar et al., 2008; Ovissipour et al., 2009; Chalamaiah et al., 2010; Foh et

416 al., 2011). The low moisture content of protein hydrolysates is related to the type of

417 sample and to the higher temperatures employed during the process of evaporation and

418 spray drying. During these processes, the sample lost most of its moisture (Bueno-

419 Solano, Lopez-Cervantes, Campas-Baypoli, Lauterio-Garcia, Adan-Bante, & Sanchez-

420 Machado, 2009).

421 The ash content of fish protein hydrolysates was reported by many studies ranged

422 between 0.45 to 27% of total composition (Bhaskar et al., 2008; Shahidi et al., 1995;

423 Benjakul & Morrissey, 1997; Sathivel et al., 2003; Sathivel et al., 2005; Wasswa et al.,

424 2007; Thiansilakul et al., 2007; Pacheco-Aguilar et al., 2008; Ovissipour et al., 2009; Yin

425 et al., 2010; Chalamaiah et al., 2010; Nilsang et al., 2005; Choi et al., 2009; Mazorra-

426 Manzano et al., 2011). The relatively high ash content of fish protein hydrolysates is due

17
427 to usage of added acid or base for adjustment of pH of medium (Liceaga-Gesualdo & Li-

428 Chan, 1999; Kristinsson & Rasco, 2000a; Gbogouri et al., 2004; Dong et al., 2005;

429 Pacheco-Aguilar et al., 2008; Choi et al., 2009).

430
431 3. Amino acid composition of fish protein hydrolysates
432
433 Protein hydrolysates obtained after hydrolysis of proteins are composed of free

434 amino acids and short chain peptides exhibiting many advantages as nutraceuticals or

435 functional foods because of their amino acid profile. Amino acid composition of any food

436 proteins has significant role in various physiological activities of human body and affects

437 either directly or indirectly in maintaining good health. Amino acids are essential for

438 synthesis of a wide variety of proteins with important functions including carriers of

439 oxygen, vitamins, CO2, enzymes and structural proteins. The amino acid composition of

440 fish protein hydrolysates is important because of the nutritional value and the influence

441 on the functional properties (Santos et al., 2011). Table 2 shows the amino acid

442 composition of protein hydrolysates prepared from various fish processing waste

443 proteins. Several authors have been described the amino acid composition of protein

444 hydrolysates produced from processing waste proteins of different fish species. These

445 include capelin (Mallotus villosus) (Shahidi et al., 1995), pacific whiting (Merluccius

446 productus) (Benjakul & Morrissey, 1997), herring (Clupea harengus) (Liceaga-Gesualdo

447 & Li-Chan, 1999; Sathivel et al., 2003), icelandic scallop (Chlamys islandica) (Mukhin et

448 al., 2001), black tilapia (Oreochromis mossambicus) (Abdul-Hamid et al., 2002), salmon

449 (Salmo salar) (Gbogouri et al., 2004), red salmon (Oncorhynchus nerka) (Sathivel et al.,

450 2005), Saurida elongate (Dong et al., 2005), tuna (Nilsang et al., 2005), round scad

451 (Decapterus maruadsi) (Thiansilakul et al., 2007), grass carp (Ctenopharyngodon idella)

18
452 (Wasswa et al., 2007), catla (Catla catla) (Bhaskar et al., 2008), silver carp

453 (Hypophthalmichthys molitrix) (Dong et al., 2008), yellow stripe trevally (Selaroides

454 leptolepis) (Klompong et al., 2009a), sole (Gimenez et al., 2009), Persian sturgeon

455 (Acipenser persicus) (Ovissipour et al., 2009), atlantic salmon; Coho salmon; alaska

456 pollack and blue whiting (Nakajima et al., 2009), cat fish (Ictalurus punctatus) (Yin et

457 al., 2010), bluewing searobin (Prionotus punctatus) (Santos et al., 2011), Misgurnus

458 anguilliacaudatus (You, Zhao, Regenstein, & Ren, 2011). Among different body parts of

459 fish, muscle protein is the most extensively studied and reported source (Klompong et al.,

460 2009a; Nakajima et al., 2009; Ghassem et al., 2011).

461 Fish protein hydrolysates have been reported to exhibit variation in their amino

462 acid composition (Wasswa et al., 2007; Bhaskar et al., 2008; Ovissipour et al., 2009). The

463 variation in amino acid composition of different fish protein hydrolysates mainly depends

464 on several factors such as raw material, enzyme source, and hydrolysis conditions

465 (Klompong et al., 2009a; Klompong, Benjakul, Kantachote, & Shahidi, 2009b). The

466 essential amino acids required for maintaining of good health have been found

467 abundantly in fish protein hydrolysates (Sathivel et al., 2003; Sathivel et al., 2005;

468 Klompong et al., 2009b; Yin et al., 2010). Among all the amino acids, aspartic acid and

469 glutamic acid were found to be higher in most of the reported fish protein hydrolysates

470 (Yin et al., 2010; Klompong et al., 2009a; Ghassem et al., 2011; Hou, Li, & Zhao,

471 2011a). Fish muscle hydrolysates contained all essential and non-essential amino acids

472 (Wasswa et al., 2007; Klompong et al., 2009a; Nakajima et al., 2009; Ghassem et al.,

473 2011; Khantaphant et al., 2011). In addition to muscle hydrolysates, head, skin and

474 visceral hydrolysates were reported to contain the all essential and non-essential amino

19
475 acids (Sathivel et al., 2005; Gimenez et al., 2009; Yin et al., 2010; Bhaskar et al., 2008;

476 Ovissipour et al., 2009), whereas aromatic amino acids were not reported in fish frame

477 protein hydrolysates (Hou et al., 2011b). Hence, the protein hydrolysates produced from

478 different parts of fish can be used as good source of essential amino acids.

479
480
481 4. Antioxidant activities of fish protein hydrolysates
482
483 Reactive oxygen species (ROS) and free radicals are generated during cellular

484 respiration in humans and other aerobic organisms. The ROS and free radicals play an

485 important role in several diseases such as neurodegenerative disorders, hypertension,

486 inflammation, cancer, diabetes, Alzheimer disease, Parkinson’s disease and ageing

487 problems (Bougatef et al., 2009; Ngo et al., 2010). The ROS and free radicals contain

488 unpaired electrons in valency shell and attract electrons from other substances, thus

489 making oxidative stress in the cells or tissues. These free radicals are unstable and react

490 rapidly with the other substances or molecules in the body, leading to cell or tissue injury.

491 In addition to the physiological production of ROS and free radicals, oxidation of fats and

492 oils in food products during processing and storage leads to production of undesirable

493 secondary lipid peroxidation products (Sarmadi & Ismail, 2010). In order to prevent lipid

494 peroxidation in food products, in food and pharmaceutical industries, many synthetic

495 antioxidants such as butylated hydroxyl toluene (BHT), butylated hydroxyl anisole

496 (BHA), tertiary butyl hydro quinone (TBHQ), and propyl gallate (PG) have been used

497 (Kim & Wijesekara, 2010). Due to the potential health risks of synthetic antioxidants the

498 search for safe natural antioxidants is important.

499
500

20
501 4.1. Production of antioxidative protein hydrolysates or peptides from fish proteins

502 An antioxidant is defined as any substance that considerably delays or inhibits the

503 oxidation of a substance. Antioxidant substances in food play significant role as health-

504 benefiting factors that protect the body from oxidative stress. In recent years, fish protein

505 sources have gained much interest as potential antioxidative peptide sources, particularly

506 due to the availability of large quantities of processing wastes and underutilized species.

507 Recently, a number of studies demonstrated that protein hydrolysates derived from fish

508 proteins are potential sources of antioxidant peptides and several antioxidant peptides

509 from these protein hydrolysates have been isolated (Jun, Park, Jung, & Kim, 2004; Je et

510 al., 2005; Je et al., 2007; Hsu, Lu, & Jao, 2009; Bougatef et al., 2010; You et al., 2010b;

511 Jai Ganesh et al., 2011; Sampath Kumar et al., 2011).

512 Several recent studies have shown that fish protein hydrolysates or peptides with

513 antioxidant activities can be released from fish proteins after enzymatic hydrolysis. These

514 antioxidative peptides are inactive within the sequence of the precursor protein molecules

515 but can be released after enzymatic hydrolysis. The antioxidative protein hydrolysates or

516 peptides can be produced from fish protein sources by using various processes such as in

517 vitro enzymatic hydrolysis, autolytic process using endogenous enzymes, microbial

518 fermentation, and simulated gastric digestion (Je et al., 2007; Rajapakse, Mendis, Jung,

519 Je, & Kim, 2005; Nazeer et al., 2011; Ren et al., 2008; Bougatef et al., 2010; Kim, Je, &

520 Kim, 2007; Phanturat et al., 2010). Among these methods, in vitro hydrolysis of fish

521 proteins using exogenous proteolytic enzymes is widely applied process for the

522 production of antioxidative fish protein hydrolysates or peptides. During hydrolysis,

523 peptide bond cleavage allows the release of active peptides capable of sequestering

21
524 oxygen radicals, chelating prooxidant metal ions and inhibiting lipid peroxidation in food

525 systems (You et al., 2010b). Selection of appropriate proteolytic enzyme is an important

526 factor for the release of antioxidant peptides from fish proteins. Proteolytic enzymes such

527 as Alcalase, a-chymotrypsin, Neutrase, papain, pepsin, trypsin, pancreatin, Flavourzyme,

528 bromelain, Pronase E, Protamex, Orientase, thermolysin, Validase, protease A amano,

529 protease N amano and cryotin F derived from plant, animal and microbial sources have

530 been successfully tested for the production of antioxidative peptides from fish protein

531 sources (Wu et al., 2003; Samaranayaka & Li-chan, 2008; Ren et al., 2008; Raghavan &

532 Kristinsson, 2008; Nakajima et al., 2009; Ngo et al., 2010; Batista et al., 2010; Hsu,

533 2010) (Table 3). Besides selection of appropriate proteolytic enzymes, the physico-

534 chemical conditions of the process such as temperature and pH for optimal activity of

535 enzyme and hydrolysis time are vital in the production of antioxidative protein

536 hydrolysates or peptides with desirable functional properties (Samaranayaka & Li-chan,

537 2011).

538
539 4.2. Isolated antioxidative peptides from fish proteins and their mechanism

540 Table 3 shows the antioxidant activities of protein hydrolysates or isolated

541 peptides derived from from different fish protein sources. The first known scientific study

542 reported the antioxidant activity of fish protein hydrolysates was in 1995 by Shahidi et al.

543 (1995). Since then a number of studies reported the antioxidative activities for various

544 fish protein hydrolysates (Yang et al., 2008; Phanturat et al., 2010; Zhong, Ma, Lin, &

545 Luo, 2011). Antioxidant peptides have been isolated from several hydrolyzed fish

546 proteins (Table 3). Several antioxidative peptides isolated from fish proteins have been

547 reported to contain 2-16 amino acid residues (Jun et al., 2004; Je et al., 2005; Je et al.,

22
548 2007; Wang et al., 2008; Kim et al., 2007; You et al., 2010b; Bougatef et al., 2010; Ngo

549 et al., 2010). Antioxidative peptides from fish proteins are considered to be safe and

550 healthy molecules with low molecular weight, easy absorption, low cost, and high

551 activity (Sarmadi & Ismail, 2010). Antioxidative properties of peptides isolated from fish

552 proteins are related to their sequence, composition and hydrophobicity (Jun et al., 2004;

553 Je et al., 2007; Ren et al., 2008, Bougatef et al., 2010). It is reported that the antioxidant

554 peptides possess some metal chelation or hydrogen/electron donating activity, which

555 could allow them to interact with free radicals and terminate the radical chain reaction or

556 prevent their formation (Ren et al., 2008; You et al., 2010b). Therefore, the amino acid

557 constituents and the sequence of the peptides are very important for their antioxidant

558 activity. It has been shown that hydrophobic amino acids and one or more residues of

559 Histidine, Proline, Methionine, Cysteine, Tyrosine, Tryptophan and Phenylalanine can

560 enhance the activities of the antioxidant peptides (Je et al., 2007; Ren et al., 2008; You et

561 al., 2010b). The presence of hydrophobic sequences in the peptides could interact with

562 lipid molecules and could scavenge by donating protons to lipid derived radicals (Je et

563 al., 2007).

564 In a study Jun et al. (2004) isolated an antioxidative peptide composed of 10

565 amino acid residues, Arg-Pro-Asp-Phe-Asp-Leu-Glu-Pro-Pro-Tyr, from yellowfin sole

566 (Limanda aspera) frame proteins. This peptide was produced by the combination of

567 mackerel intestinal crude enzyme and pepsin. In another study, Je et al. (2005) identified

568 an antioxidative peptide, Leu-Pro-His-Ser-Gly-Tyr (672 Da), from Alaska pollack

569 (Theragra chalcogramma) frame protein hydrolysate that was prepared with mackerel

570 intestine crude enzyme and reported that the isolated peptide showed 35% scavenging

23
571 activity on hydroxyl radicals at 53.6 µM concentration. Kim et al. (2007) employed six

572 proteases (pepsin, trypsin, papain, α-chymotrypsin, Alcalase and Neutrase) to extract

573 antioxidative peptides from hoki (Johnius belengerii) frame proteins. Among

574 hydrolysates, peptic hydrolysate having the highest antioxidant activity was further

575 separated into four groups using ultrafiltration membranes and purified 1801 Da peptide,

576 Glu-Ser-Thr-Val-Pro-Glu-Arg-Thr-His-Pro-Ala-Cys-Pro-Asp-Phe-Asn, from peptic

577 hydrolysates. This purified peptide efficiently inhibited lipid peroxidation and was a

578 potent free radical scavenger. Je et al. (2007) identified antioxidant peptide, Val-Lys-Ala-

579 Gly-Phe-Ala-Trp-Thr-Ala-Asn-Gln-Gln-Leu-Ser (1519 Da) from peptic hydrolysate of

580 tuna backbone protein and reported that the isolated peptide showed significant inhibition

581 of lipid peroxidation in linoleic acid emulsion system and quenching of free radicals

582 (DPPH, hydroxyl and superoxide) in a dose-dependent manner. Ren et al. (2008) isolated

583 a potent antioxidant peptide, Pro-Ser-Lys-Tyr-Glu-Pro-Phe-Val (966.3 Da), from grass

584 carp muscle protein hydrolysate that was produced by Alcalase. It has been shown that

585 basic peptides had greater capacity to scavenge hydroxyl radical than acidic or neutral

586 peptides.

587
588 Two aromatic amino acids, tyrosine and phenylalanine, are known to act

589 positively as direct radical scavengers. The antioxidant activity of tyrosine is due to the

590 special capability of phenolic groups to serve as hydrogen donors (Ren et al., 2008; Jun et

591 al., 2004). The amino acids such as histidine (His), methionine (Met) and cysteine (Cys)

592 are very important to the radical scavenging activity of peptides due to their special

593 structural characteristics: the imidazole group in His has the proton-donation ability; Met

24
594 is prone to oxidation of the Met sulfoxide; Cys donates the sulfur hydrogen (Ren et al.,

595 2008; Hsu et al., 2009; Bougatef et al., 2010).

596
597 The exact mechanism of action of peptides as antioxidants has not clearly been

598 understood, but some amino acids such as His, Met, Cys, Pro, Val, Phe, Tyr, Trp have

599 been reported to contain vital role in the antioxidative activities of peptides or protein

600 hydrolysates generated from fish proteins (Ren et al., 2008; Hsu et al., 2009; You et al.,

601 2009; Bougatef et al., 2010; Sarmadi & Ismail, 2010; Samaranayaka & Li-chan, 2011).

602 The antioxidant potential of protein hydrolysates depend on amino acid composition and

603 on disruption of tertiary structure of parent proteins by enzymatic hydrolysis that results

604 in increasing the solvent accessibility of oxidatively labile amino acid residues (Elias,

605 Kellerby, & Decker, 2008). Extensive enzymatic hydrolysis of proteins results in

606 decreased antioxidant activity due to free amino acids. Protein hydrolysates (peptides) are

607 potential antioxidants than free amino acids, due to their chemical composition and

608 physical properties (Elias et al., 2008).

609
610 Wang, Zhu, Han, and Wang (2008) identified an antioxidative dipeptide, Pro-Arg,

611 from Salmon protamine hydrolysate. This identified peptide showed hydroxyl radical-

612 scavenging activity of 21.04% at a low concentration of 40 µg/mL. In a study conducted

613 by Hsu et al. (2009), the isolation of three antioxidative peptides was reported from tuna

614 cooking juice hydrolysates prepared by Orientase. These three peptides comprised 4–10

615 amino acids sequences, and the structures of the peptides were Pro-Val-Ser-His-Asp-His-

616 Ala-Pro-Glu-Tyr (1305 Da), Pro-Ser-Asp-His-Asp-His-Glu (938 Da), and Val-His-Asp-

617 Tyr (584 Da). The authors demonstrated that the sequence of two antioxidative peptides

25
618 contained histidine and proline residues, and the prolyl polypeptides are sensitive to

619 oxidation by Cu(II)/H2O2 and generate mainly β-aminobutyric acid, hydro-oxyproline,

620 aspartic acid, and glutamic acid after hydrolysis of the oxidized substrates. In another

621 investigation by Hsu (2010) tuna dark muscle by-product was hydrolyzed using Orientase

622 and protease XXIII and identified two antioxidative peptides, Leu-Pro-Thr-Ser-Glu-Ala-

623 Ala-Lys-Tyr (978 Da) and Pro-Met-Asp-Tyr-Met-Val-Thr (756 Da). The hydrophobic

624 property of antioxidative peptides exerts high affinity to linoleic acid and it is important

625 to inhibit lipid peroxidation (Hsu, 2010). The peptides containing 5–16 amino acid

626 sequences are also responsible for the strong inhibition activities on the autooxidation of

627 linoleic acid (Hsu, 2010). You et al. (2010b), purified 464.2 Da antioxidant peptide, Pro-

628 Ser-Tyr-Val, from loach (Misgurnus anguillicaudatus) protein hydrolysate produced by

629 papain, and reported that the isolated peptide showed 9.14-fold higher scavenging

630 activity for hydroxyl radical than crude hydrolysate. A study performed by Ngo et al.

631 (2010) investigated the antioxidant activities of a peptide isolated from Nile tilapia

632 (Oreochromis niloticus) scale gelatin Alcalase-derived hydrolysate. The active peptide

633 was identified as Asp-Pro-Ala-Leu-Ala-Thr-Glu-Pro-Asp-Pro-Met-Pro-Phe (1382.57 Da)

634 and reported that purified peptide showed no cytotoxic effect on mouse macrophages

635 (RAW 264.7) and human lung fibroblasts (MRC-5). In addition, the isolated peptide

636 scavenged hydroxyl, DPPH and superoxide radicals at the IC50 values of 7.56, 8.82 and

637 17.83 µM, respectively. Bougatef et al. (2010) reported seven antioxidative peptides such

638 as Leu-His-Tyr, Leu-Ala-Arg-Leu, Gly-Gly-Glu, Gly-Ala-His, Gly-Ala-Trp-Ala, Pro-

639 His-Tyr-Leu and Gly-Ala-Leu-Ala-Ala-His from sardinelle (Sardinella aurita) industrial

640 wastes generated with crude enzyme extract from sardine (Sardina pilchardus). They

26
641 reported that the first tripeptide displayed the highest DPPH radical-scavenging activity

642 (63±1.57% at 150 µg/ml) and, the presence of amino acids His and Tyr sequence could

643 contribute significantly to the antioxidant activity of the peptides. The antioxidant

644 activities of histidine-containing peptides have the chelating and lipid radical trapping

645 ability due to the imidazole ring (You et al., 2009; Hsu et al., 2009; Bougatef et al.,

646 2010). Jai Ganesh et al. (2011) reported the sequence of antioxidative peptide as Ala-

647 Met-Thr-Gly-Leu-Glu-Ala with a mass of 701.9 Da isolated from black pomfret

648 (Parastromateus niger) visceral waste and also reported the protection ability of this

649 peptide toward hydroxyl radical-induced oxidative DNA damage and inhibition of lipid

650 peroxidation. Sampath kumar et al. (2011) isolated two antioxidant peptides such as Asn-

651 His-Arg-Tyr-Asp-Arg (856 Da) and Gly-Asn-Arg-Gly-Phe-Ala-Cys-Arg-His-Ala

652 (1101.5 Da) from skin protein hydrolysates of horse mackerel (Magalaspis cordyla) and

653 croaker (Otolithes ruber) respectively. These two isolated peptides have shown higher

654 activity against polyunsaturated fatty acid (PUFA) peroxidation than the natural

655 antioxidant α-tocopherol.

656 Apart from isolated antioxidative peptides from fish protein hydrolysates, several

657 recent studies have also reported the antioxidative activities for crude protein

658 hydrolysates produced from various fish protein sources (Khantaphant & Benjakul, 2008;

659 Gimenez et al., 2009; Yang et al., 2009; Zhuang et al., 2009; Jia, Zhou, Lu, Chen, Li, &

660 Zheng, 2010; Picot et al., 2010; Aleman et al., 2011; Sampath Kumar et al., 2011). Table

661 3 lists the antioxidative activities reported for fish protein hydrolysates and enzymes used

662 for their production. Fish muscle contains an excellent amino acid composition and is an

663 excellent source of nutritive and easily digestible proteins. However, many fish species

27
664 are underutilized because of their extreme perishable nature and other inherent problems

665 related to undesirable odour, texture and taste (Kristinsson & Rasco, 2000b). Muscle

666 proteins from various fish species have been successfullly tested for production of protein

667 hydrolysates with different antioxidative activities (Table 3) (Thiansilakul et al., 2007;

668 Klompong, Benjakul, Kantachote, Hayes, & Shahidi, 2008; Raghavan & Kristinsson,

669 2008; Raghavan, Kristinsson, & Leeuwenburgh, 2008; Dong et al., 2008; Ren et al.,

670 2008; Bougatef et al., 2009; Chabeaud et al., 2009; Nakajima et al., 2009; Hsu, 2010;

671 Rajaram & Nazeer, 2010; Nalinanon et al., 2011; Naqash & Nazeer, 2011a). In addition

672 to fish muscle proteins, other protein sources from fish such as backbones of Tuna (Je et

673 al., 2007), Gadus morhua (Slizyte et al., 2009), seela (Sphyraena barracuda) and ribbon

674 fish (Lepturacanthus savala) (Nazeer et al., 2011), Exocoetus volitans (Naqash & Nazeer,

675 2011b) have also been used for the production of antioxidant protein hydrolysates.

676 Protein hydrolysates obtained from Tuna liver are reported to exhibit the antioxidant

677 properties (Je et al., 2009; Ahn et al., 2010). Antioxidant activities have also been

678 reported for other by-product waste from fish industry including visceral waste proteins

679 and fish head proteins (Souissi et al., 2007; Batista et al., 2010; Bougatef et al., 2010;

680 Barkia, Bougatef, khaled, & Nasri, 2010; Jai Ganesh et al., 2011; Hathwar et al., 2011).

681
682 The antioxidant protein hydrolysates derived from various fish proteins have

683 potential for nutritional, pharmaceutical, cosmetic and nutraceutical application as

684 functional ingredient due to their health promoting effects. In addition, the search for

685 natural antioxidants for human consumption as safe alternatives to synthetic antioxidants

686 (BHT, BHA, tertiary butylhydroquinone and propyl gallate) is important for the food

687 industry. The antioxidative protein hydrolysates derived from fish proteins could be used

28
688 as natural antioxidants as replacement for synthetic antioxidants (Hsu, 2009; Bougatef et

689 al., 2010). However, before being used for human consumption further studies are

690 required to evaluate the effectiveness of these protein hydrolysates in animals and human

691 subjects.

692

693 5. Potential applications of fish protein hydrolysates

694
695 Protein hydrolysates generated from fish proteins are good nutritional

696 supplements as bioactive compounds and can be easily absorbed and utilized for various

697 metabolic activities (Nesse, Nagalakshmi, Marimuthu, & Mamta singh, 2011). Currently,

698 there is a resurgence of interest in the use of natural bioactive products (functional

699 food/nutraceutical) among the general public, with many healthy subjects and patients

700 taking them for prevention and treatment of multiple conditions including gastrointestinal

701 disorders (Marchbank, Limdi, Mahmood, Elia, & Playford, 2008). In many countries,

702 traditional and commercial preparations of fish protein hydrolysates are currently used as

703 health food/functional foods/nutraceuticals. Table 4 lists the brand names, particulars and

704 health promoting applications of commercially available nutraceuticals in different

705 countries prepared from fish protein hydrolysates.

706 Fish protein hydrolysates have potential application as functional ingredients in

707 different foods because they possess numerous important and unique properties such as

708 water holding capacity, oil absorption capacity, protein solubility, gelling activity,

709 foaming capacity and emulsification ability (Chalamaiah et al., 2010). Fish protein

710 hydrolysates (FPH) have been successfully tested for incorporation into different food

711 systems such as cereal products, fish and meat products, desserts and crackers etc.

29
712 (Kristinsson & Rasco, 2000). Only limited numbers of clinical trails were conducted on

713 FPHs to test their efficacy as functional food/health food in humans. Marchbank et al.

714 (2008) studied the small intestinal injury (induced by indomethacin) protective effect of a

715 commercial fish protein hydrolysate, which is marketed in the US as an “over the

716 counter” health food supplement, derived from the controlled proteolytic yeast

717 fermentation of pacific whiting (Merluccius productus). In this study, double-blind,

718 placebo-controlled, crossover clinical trial was conducted using eight human volunteers

719 at a clinically relevant dose of indomethacin (50 mg t.d.s. p.o. for 5 days) with 7 days of

720 fish hydrolysate or placebo starting 2 days prior to indomethacin. It is found that

721 dyspeptic symptoms occurred in four of eight subjects taking indomethacin alone, but

722 zero of eight subjects when fish hydrolysate was co-administered. Recently in another

723 study Nesse et al. (2011) investigated the effect of fish protein hydrolysate (Amizate,

724 prepared from salmon fish protein) on 438 Indian malnourished children (6–8 years) of

725 Grade I and II (Gomez’s classification) with respect to immunoglobulins, CD4/CD8

726 ratios and hemoglobin. In this study, authors randomly grouped the children (438) into

727 three groups, group A was administered 3 g of Amizate in chocolate drink of 120 ml,

728 while group B was given 6 g of Amizate in chocolate drink of 120 ml and the children

729 grouped as group C were given plain chocolate drink of 120 ml without Amizate. The

730 authors reported that this user trial study (at the beginning and the end after 4 months)

731 indicated that the levels of the immunological parameters were not significantly altered

732 by the fish protein hydrolysate treatment, but the values of immunoglobulins and

733 CD4/CD8 ratios of malnourished children (India) are in the normal range and were in

734 accordance with the reported values of various ethnic groups.

30
735 Fish protein hydrolysates (FPHs) have been used in aquaculture feeds in order to

736 enhance the growth and survival of fish (Kotzamanis, Gisbert, Gatesoupe, Infante, &

737 Cahu, 2008). Kotzamanis et al. (2008) incorporated two fish protein hydrolysates, from

738 Sardina pilchardus, into four diets prepared for start-feeding sea bass larvae, at two

739 different levels (10% and 19% of total ingredients), and reported that the peptides in the

740 protein hydrolysates affected the growth performance and immunological status of sea

741 bass larvae. In another study, a pollock protein hydrolysate was used for enrichment of

742 the live feed offered to halibut larvae from the onset of exogenous feeding and studied

743 the effects of treatment on selected innate immune parameters and concluded that feeding

744 peptide-enriched live feed to larvae stimulated the production of lysozyme and C3 during

745 the first weeks in feeding (Hermannsdottir, Johannsdottir, Smaradottir, Sigurgisladottir,

746 Gudmundsdottir, & Bjornsdottir, 2009). Nguyen, Perez-Galvez, and Berge (2012)

747 conducted a feeding trial to evaluate the effect of the supplementation of hydrolysates

748 from tuna head on the survival and growth of shrimp Penaeus vannamei and reported the

749 hydrolysates of tuna head improved significantly both growth and the survival rates of

750 shrimps.

751 FPHs can be used as an excellent source of nitrogen for maintaining the growth of

752 different microorganisms. Ghorbel, Souissi, Triki-Ellouz, Dufosse, Guerard, and Nasri

753 (2005) used various fish protein hydrolysates (FPH) from sardinella (Sardinella aurita)

754 as nitrogen sources for the production of extracellular lipase by the filamentous fungus

755 Rhizopus oryzae and reported that the best results were obtained with defatted fish

756 protein hydrolysates. In another study by Safari, Motamedzadegan, Ovissipour,

757 Regenstein, Gildberg, and Rasco (2009) the hydrolysates generated from yellowfin tuna

31
758 (Thunnus albacares) head waste have been shown to be effective in promoting the

759 growth of lactic acid bacteria better than the commercial MRS media.

760 Fish protein hydrolysates can also be used as cryoprotective agents. A study

761 conducted by Cheung, Liceaga, and Li-Chan (2009) reported the cryoprotective ability of

762 protein hydrolysates produced by proteolysis of Pacific hake (Merluccius productus) with

763 Alcalase and Flavourzyme. In this study, the authors investigated Pacific hake protein

764 hydrolysates as a potential alternative to the 1:1 blend of sucrose-sorbitol commonly used

765 for cryoprotection of frozen fish mince, and results obtained provided a strong evidence

766 to support development of FPH as a new generation cryoprotectant to maintain quality of

767 frozen fish.

768
769 Fish protein hydrolysates have been tested successfully for their potential

770 application as antioxidant agents. Dekkers et al. (2011) investigated the oxidative

771 stability of mahi mahi red muscle dipped in tilapia protein hydrolysates for 2 or 4 minutes

772 (stored at 4oC) and reported that dip treatments significantly decreased (p<0.05) the

773 formation of lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS)

774 over 90 h storage time. In a recent study, Khaled et al. (2012) studied the effect of protein

775 hydrolysates from sardinelle (Sardinella aurita) on the oxidative status and blood lipid

776 profile of cholesterol-fed rats. Authors reported that the treatment of

777 hypercholesterolemic diet rats with sardinelle protein hydrolysates reduced the

778 malondialdehyde (MDA) concentration and increased the antioxidant enzyme

779 (superoxide dismutase, glutathione peroxidase and catalase) activities and high density

780 lipoprotein (HDL) cholesterol. Apart from demonstrated in vitro and in vivo antioxidant

781 activities, it is reported that fish protein hydrolysates can have the ability to alter the lipid

32
782 metabolism in experimental animals (Wergedahl et al., 2004; Khaled et al., 2012).

783 Wergedahl et al. (2004) showed the protein hydrolysate from salmon reduces the plasma

784 total cholesterol and increases the proportion of HDL cholesterol in liver of Zucker rats.

785 This study clearly demonstrated the potential usefulness of FPH as a cardioprotective

786 nutrient. Saito, Kiyose, Higuchi, Uchida, and Suzuki, (2009) have demonstrated the

787 efficacy of collagen hydrolysates from salmon and trout skins on the lipid profile in rats

788 and reported that the hydrolysates lowered the levels of plasma total lipids and

789 triglycerides compared to control group rats. In another investigation, Hosomi, Fukao,

790 Fukunaga, Okuno, and Yagita, (2010) reported the alaska pollock (Theragra

791 chalcogramma) fillet protein hydrolysates decreased the serum and liver cholesterol

792 contents of experimental rats through the enhancement of fecal acidic and neutral

793 excretions. Besides animal studies, more number of investigations is required in humans

794 to prove FPHs efficacy and safety.

795

796 6. Conclusions

797 In this review, the proximate composition, amino acid composition and

798 antioxidant activities and potential applications of fish protein hydrolysates are presented.

799 Currently, testing of bioactivity of fish protein hydrolysate is restricted to in vitro

800 experiments and only few investigations were carried out in animals and humans. The

801 fate of fish protein hydrolysates in the gastrointestinal tract, their absorption, and

802 bioavailability are the major questions to be answered before exploitation for human

803 nutrition as functional foods. The future research on FPHs would be very bright since the

804 FPHs market is expanding enormously all over the world.

33
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1247

53
 Table 1 Proximate composition of fish protein hydrolysates from various fish sources
Fish Species and particulars Protein (%) Fat (%) Moisture (%) Ash (%) Reference
Raw A 87.9 4.0 4.7 12.5
Herring P 85.3 4.7 4.8 9.6
Clupea harengus Herring A 82.3 3.7 3.9 13.3
Hoyle and Merritt
protein hydrolysates press cake P 83.4 3.6 3.2 9.9 (1994)
Ethanol A 83.7 1.8 3.3 12.0
extracted
Herring P 85.7 0.9 3.9 7.5
Mallotus villosus Alcalase 72.4±0.70 0.18±0.03 6.34±0.11 20.8±1.82
protein hydrolysates papain 78.3±1.92 0.39±0.02 5.32±0.24 17.7±1.80 Shahidi et al. (1995)
Neutrase 71.2±0.83 0.21±0.02 5.26±0.11 21.5±0.53
Merluccius productus Benjakul and Morrissey
82.25±0.05 3.94±0.13 - 13.82±0.08
solid waste protein hydrolysates (1997)
Clupea harengus Liceaga-Gesualdo and
77.0±0.2 0.77±0.1 3.98±0.02 21.7±0.1
protein hydrolysates Li-Chan (1999)
Salmo salar Kristinsson and Rasco
88.39±0.55 0.23±0.12 0.92±0.22 8.96±0.13
muscle protein hydrolysates (2000a)
processing waste
81.3 0.56 5.55 12.6
IChlamys islandica hydrolysate
Mukhin et al. (2001)
pancreatic fish-
73.1 0.80 8.67 17.4
meal hydrolysates
Oreochromis mossambicus type A 49.6±0.19 2.80±0.13 3.93±0.15 8.65±0.01
Abdul-Hamid et al.
protein hydrolysates
type B 37.7±0.05 2.56±0.10 1.58±0.12 8.56±0.03 (2002)
HBH 87±0.2 0.4±0.1 3.0±0.1 10.1±0.1
Clupea harengus
HHH 85.2±0.4 1.2±0.2 3.5±0.2 10.1±0.3 Sathivel et al. (2003)
by-product hydrolysates
HGH 77±0.4 1.5±0.2 6.2±0.3 15.3±0.1
Salmo salar 82.3±1.9
0.8±0.02 5.3±0.2 10.4±1.1 Gbogouri et al. (2004)
head protein hydrolysates
Tuna
66.40±0.27 2.37±0.52 7.25±0.09 25.94±0.04 Nilsang et al. (2005)
by-product protein hydrolysates
Saurida elongate
84.7 3.5 8.5 7.1 Dong et al. (2005)
protein hydrolysates
Alcalase 63.3±2.1 23.7±2.2 5.9±0.4 7.1±0.5
Flavourzyme 500L 62.8±0.4 24.5±0.6 5.0±0.1 7.7±0.2

Oncorhynchus nerka Palatase 2000L 62.3±0.9 23.9±1.4 6.1±0.3 7.7±0.4

Sathivel et al. (2005)
head protein hydrolysates protex 6L 63.6±0.7 23.1±0.6 6.2±0.4 7.1±0.1
GC 106 64.8±0.3 22.6±0.5 5.6±0.3 7.2±0.1
Neutrase 64.8±4.7 22.5±5.7 5.7±0.5 6.9±0.6
Decapterus maruadsi Thiansilakul et al.
69.0±3.57 0.15±0.03 8.75±0.40 24.56±2.56
protein hydrolysates (2007)
Ctenopharyngodon idella
92.0±1.75 0.2±0.10 2.87±0.05 5.86±0.60 Wasswa et al. (2007)
skin protein hydrolysates

54
 Table 1 continued
Fish Species and particulars Protein (%) Fat (%) Moisture (%) Ash (%) Reference
Sardinella aurita FPH1 75.01±1.72 8.53±1.11 1.35±0.55 14.81±0.14
by-product protein FPH2 72.99±1.82 10.21±1.58 2.83±0.42 13.06±0.13
Souissi et al. (2007)
hydrolysates
FPH3 73.05±1.91 10.29±1.76 4.56±0.27 12.10±0.12
Catla catla
13.85±1.55* 1.94±0.35 3.85±0.65 0.45±0.15 Bhaskar et al. (2008)
visceral protein hydrolysates
Merluccius productus 10% DH 88.6±0.3 0.1±0.1 3.6±1.9 11.9±0.1
Pacheco-Aguilar et al.
muscle protein 15% DH 88.4±0.3 0.2±0.2 3.2±0.1 11.7±0.4 (2008)
hydrolysates 20% DH 85.6±0.3 0.3±0.1 2.8±0.8 11.9±0.4
Acipenser persicus
65.82±7.02 0.18±0.4 4.45±0.67 7.67±1.24 Ovissipour et al. (2009)
visceral protein hydrolysate
PTPH 85.9±0.6 - 4.4±0.7 4.7±0.2
Pennahia argentata PTSH 85.4±0.6 - 4.4±0.7 8.4±0.4
Choi et al. (2009)
protein hydrolysates FTPH 83.9±0.6 - 3.6±0.0 3.7±0.1
FTSH 80.7±0.9 - 5.2±0.3 8.4±0.3
Cirrhinus mrigala Alcalase 85.0±2.32 6.1±0.20 5.2±0.15 3.7±0.08 Chalamaiah et al.
egg protein hydrolysates papain 70.0±1.9 14.9±0.5 8.2±0.6 6.9±0.2 (2010)
Ictalurus punctatus CSSH 88.34±0.21 1.83±0.35 6.75±0.11 3.07±0.18
Yin et al. (2010)
skin protein hydrolysates CSISH 39.73±1.38 52.28±1.60 6.10±0.17 1.89±0.20
Lutjanus vitta muscle HAP 87.36±0.12 0.61±0.06 11.16±0.39 1.76±0.73 Khantaphant et al.
protein hydrolysates HFP 86.55±0.19 0.64±0.04 11.96±0.52 1.78±0.32 (2011)
Merluccius productus Mazorra-Manzano et al.
85.6±2.3 0.3±0.1 2.5±0.6 16.6±0.3
muscle protein hydrolysates (2011)
Oreochromis niloticus FMMH 97.57±0.12 0.67±0.04 1.2±0.02 2.25±0.13
Foh et al. (2011)
meat hydrolysates HWDH 85.40±0.62 1.24±0.05 3.17±0.04 9.85±0.14
* Nitrogen content; DH= Degree of hydrolysis; FPH= Fish Protein Hydrolysates; A=Alcalase; P=Papain;
CSSH= Catfish Skin Soluble Hydrolysates; CSISH= Catfish Skin Insoluble Hydrolysates; HBH; Herring
Body Hydrolysates, HHH; Herring Head Hydrolysates, HGH; Herring Gonad Hydrolysates;
PTPH=Protamex Treated Precipitate Hydrolysates; PTSH= Protamex Treated Supernantant Hydrolysate;
FTPH= Flavourzyme Treated Precipitate Hydrolysates; FTSH= Flavourzyme Treated Supernatant
Hydrolysates; HAP=Alcalase/pyloric caeca protease treated hydrolysates; HFP= Flavourzyme/ pyloric
caeca protease treated hydrolysates; FMMH= Fresh minced meat hydrolysates; HWDH= Hot water dip
hydrolysate

55
 Table 2 Amino acid composition of various fish protein hydrolysates prepared from different fish species

Capelin Pacific whiting Herring Protein hydrolysates Black Tilapia Herring (Clupea Salmo salar Protein
Nile tilapia
(Mallotus (Merluccius (Clupea from (Oreochromis harengus) byproduct HPHs (m hydrolysat
(Oreochromus
villosus) productus) harengus) Icelandic Scallop mossambicus) protein hydrolysates mol/g es from
niloticus)
protein solid waste protein (Chlamys islandica) hydrolysates (mg/g protein) protein) Tuna
myofibrillar
hydrolysates protein hydrolysates (%) (Mukhin et al., (mg/g crude protein) (Sathivel et al., 2003) (Gbogouri byproduct
Amino acid (%) hydrolysates (g/100 g) 2001) (Abdul-Hamid et al.,
protein
et al., 2004) (g/100g)
hydrolysates
(Shahidi et al., (%) (Liceaga- 2002) (Nilsang et
(g/kg) (Candid
1995) (Benjakul & Gesualdo & Li- al., 2005)
& Sgarbieri,
Morrissey, Chan, 1999) PWH PFMH Type A Type B HBH HHH HGH
2003)
1997)
Aspartic
9.89±0.53 10.10 10.72 4.116 0.944 67.2±5.30 24.5±2.41 93.8 89.2 86.6 - 0.61 0.67
acid/Asparagine
Threonine 4.56±0.03 5.12 4.74 0.856 0.682 30.2±6.89 13.2±8.13 39.3 40.4 46.7 52 0.34 0.74
Serine 4.24±0.10 5.33 4.87 0.941 0.710 24.0±0.10 18.3±1.24 41.1 45.3 44.6 - 0.37 0.67
Glutamic acid/
13.4±0.03 13.80 15.87 2.678 1.714 151±7.05 74.5±8.28 163.4 145.1 161.3 - 0.75 1.52
Glutamine
Proline 3.67±0.03 6.00 4.54 0.741 0.300 22.6±2.98 21.8±2.02 41.0 58.9 44.7 - 0.54 1.45
Glycine 5.14±0.01* 7.88 7.59 1.600 0.248 40.9±1.66 19.6±4.05 69.8 95.0 84.5 - 1.10 2.98
Alanine 6.00±0.01 6.53 7.74 2.030 1.528 37.7±2.01 30.4±1.05 70.1 69.4 52.9 - 0.82 1.33
Valine 5.77±0.01 4.72 4.34 1.681 1.092 31.9±0.56 21.1±0.78 44.7 45.7 52.5 53 0.26 0.56
Methionine 2.05±0.01 3.02 4.94 0.457 0.400 27.6±3.01 31.7±1.09 33.3 33.1 32.8 24 0.18 0.48
Isoleucine 4.25±0.04 4.30 3.15 - - 33.6±1.26 16.6±4.67 33.2 32.6 39.5 49 0.20 0.35
Leucine 7.6O±0.00 7.16 8.42 - - 68.9±0.11 58.1±1.24 78.8 71.2 77.0 84 0.40 0.77
Tyrosine 2.47±0.06 3.50 2.64 1.909 1.566 25.8±1.08 13.5±5.07 26.3 27.4 38.8 35 0.11 0.40
Phenylalanine 3.19±0.00 3.80 3.39 2.811 2.052 59.1±2.81 41.6±2.58 34.9 37.2 42.8 44 0.16 0.64
Histidine 2.09±0.02 2.10 1.22 0.229 0.632 - - 26.0 18.9 23.8 23 0.10 2.06
Hydroxylysine 0.17±0.02 - - - - - - - - - - - -
Lysine 8.49±0.06 8.33 8.46 3.778 2.704 75.9±1.11 28.7±4.59 106.6 80.4 65.5 92 0.11 0.54
Arginine 5.70±0.02 7.29 7.06 4.611 2.406 49.1±0.50 26.9±0.23 70.5 73.0 96.3 0.33 2.76
Tryptophan 0.43±0.0 1 0.14 - 0.812 0.566 - - - - - 14 - -
Cysteine 1.34±0.00 0.92 0.30 - - - - 11.2 11.1 5 8 - -
Cystine - - - - - - - - - - - - 0.05
PWH=Processing Waste Hydrolysate; PFMH=Pancreatic Fish Meal Hydrolysate; HBH= Herring Body Hydrolysates; HHH= Herring Head Hydrolysates;
HGH= Herring Gonad Hydrolysates; * Glucine

56
 Table 2 continued
Protein Red Salmon (Oncorhynchus nerka) head Round scad Grass carp Silver carp (Hypophthalmichthys molitrix) Protein Persian
hydrolysate hydrolysates (mg of amino acid /gm protein) (Decapterus (Ctenophary Protein hydrolysates (g/100 g protein) hydrolysate sturgeon
from (Sathivel et al., 2005) maruadsi) ngodon (Dong et al., 2008) from visceral visceral
(Saurida protein idella) skin waste proteins protein
elongate) (g hydrolysates protein of Catla (Catla hydrolys
Amino acid
/kg crude (%) hydrolysates catla) (g/100g ates
protein) (Thiansilaku (g/100g protein) (g/100g)
(Dong et al., l et al., protein) (Bhaskar et al., (Ovissip
GC Alcalase Flavourzyme
2005) A F P Pr N 2007) (Wasswa et 2008) our et al.,
106 al., 2007) 0.25h 1.5h 4.0h 0.25h 1.5h 4.0h 2009)
Aspartic
89.4 88.3 87.7 87.2 89.3 94 91.1 2.04 5.54 9.86 9.03 9.94 10.35 9.87 8.85 8.63 8.3
acid/Asparagine
Threonine - 41.9 42.1 42.6 43.9 46.3 44.0 5.09 2.03 4.35 3.56 4.04 4.54 3.92 3.93 4.12 3.5
Serine 31.7 46.1 46.2 46.4 46.1 45.7 44.2 8.16 3.14 3.98 4.02 4.08 4.30 4.07 4.09 4.04 4.2
Glutamic acid/
14.6 135.1 134.9 133.3 132.5 138.5 135.6 3.47 9.44 15.65 16.1 14.75 18.69 17.7 17.43 14.71 13.7
Glutamine
Proline 52.4 65.0 66.8 66.6 64.8 53.1 58.2 0.51 8.34 7.37 7.60 8.01 4.85 4.76 5.20 6.41 3.46
Glycine 51.2 97.6 102.9 105.3 99.6 72.4 85.2 1.49 17.1 4.46 4.07 4.63 4.71 4.87 4.45 11.25 5.4
Alanine 52.6 68.4 68.8 69.6 69.2 66.3 67.7 5.31 15.6 6.82 7.13 6.02 7.92 7.67 8.49 7.16 6.3
Valine 42.8 50.1 45.2 45.5 48 51.4 50.2 6.77 2.26 7.03 7.41 7.53 5.67 6.06 5.97 4.93 5.79
Methionine 24.8 28.8 29.4 28.9 28.6 30.3 30.0 4.51 1.54 0.94 0.98 0.86 0.47 0.53 0.72 2.00 10.3
Isoleucine 39.0 37.1 36.6 35.7 37.4 42.4 39.9 3.15 1.60 4.19 4.20 4.35 3.57 3.76 3.76 3.40 3.8
Leucine 65.7 66.9 66.5 66.0 65.7 77.1 72.0 10.1 2.86 8.18 8.41 8.61 8.69 8.29 8.81 7.00 7.13
Tyrosine 27.1 32.5 30.9 31.0 32.1 36.2 34.8 5.20 3.78 3.04 2.82 2.96 2.13 2.46 2.40 2.38 2.34
Phenylalanine 59.5 40.7 39.7 39.6 39.3 44.5 42.0 4.52 2.18 3.89 4.35 4.75 3.21 3.25 3.43 3. 15 3.14
Histidine - 23.8 23.0 23.8 24.7 27.7 26.6 11.2 5.95 9.09 9.44 8.30 9.32 9.50 9.51 2.00 2.08
Hydroxyproline - 23.5 25.6 26.6 23.4 12.4 17.0 - - - - - - - - - -
Lysine 61.2 73.9 72.5 71.4 76.1 83.2 81.6 13.9 3.44 2.26 2.38 2.34 2.35 2.15 2.34 6.98 6.8
Arginine 42.4 67.7 69.2 68.4 69.2 66.8 69.6 14.0 7.22 6.09 6.14 5.89 5.32 6.03 5.40 11.82 7.28
Tryptophan - - - - - - - - - - - - - - - - -
Cysteine - - - - - - - 0.69 1.86 - - - - - - - -
Cystine - - - - - - - - - 2.70 2.38 2.95 3.92 5.06 5.23 0.41 -

A= Alcalase; F = Flavourzyme 500L; P= Palate 200L; Pr=Protex; N= Neutrase; h= hour

57
 Table 2 continued
Fish muscle hydrolysates (mg/100g dry sample) Gelatin hydrolysates Yellow Stripe Trevally
(Nakajima et al., 2009) from skin of sole (Selaroides leptolepis)
(No. of protein hydrolysates (%)
Amino acid Atlantic salmon Coho salmon Alaska pollack Blue whiting
residues/1000 (Klompong et al., 2009a)
residues) (Gimenez
PH PPH TH PH PPH TH PH PPH PH PPH
et al., 2009)
Alcalase Flavourzyme
Aspartic
0.4±0.2 11.6±1.1 12.4±0.5 1.4±0.2 17.8±1.3 15.8±2.0 11.8±07 16.9±5.3 5.9±1.2 1.1±0.6 45* 9.55 9.40
acid/Asparagine
Threonine 2.9±0.2 6.0±0.3 17.8±0.8 2.8±0.1 7.3±0.4 36.4±1.2 13.5±0.2 24.2±6.0 4.9±0.4 2.8±0.9 21 5.35 5.40
Serine 4.8±0.2 6.4±0.3 21.8±0.6 6.1±0.1 8.3±0.4 37.9±2.1 18.3±1.1 6.2±1.4 7.6±1.1 0.6±0.4 44 5.21 5.15
Glutamic acid/
3.1±0.1 29.0±1.2 54.2±5.4 6.4±0.2 45.9±2.2 45.6±1.5 3.1±0.1 29.0±1.2 32.9±5.5 5.8±1.7 73** 13.77 13.89
Glutamine
Proline - - - - - - - - - - 110 3.81 3.84
Glycine 13.7±0.8 15.2±1.0 33.0±2.7 28.3±0.1 26.0±3.6 67.4±8.0 36.7±1.2 55.4±11.6 19.1±2.5 19.0±3.7 360 8.87 8.65
Alanine 24.7±1.5 35.5±0.9 95.0±5..3 26.7±0.6 35.9±2.8 133.0±8.0 37.2±1.2 58.9±13.5 36.9±5.3 10.4±1.9 124 9.49 9.46
Valine 2.5±0.1 15.2±0.3 18.4±1.7 2.3±0.2 20.1±3.2 56.7±6.4 5.1±0.2 20.0 ± 5.2 5.4±0.8 10.3±2.2 16 3.61 3.38
Methionine 1.3±0.1 11.8±0.4 31.9±1.6 1.9±0.1 14.2±0.6 66.3±8.0 5.8±0.4 21.6±4.9 5.5±1.0 17.6±3.3 12 2.58 1.87
Isoleucine 1.2±0.1 21.9±2.2 - 1.1±0.1 17.4±4.7 68.5±1.9 3.4±0.1 17.4±3.9 4.4±0.7 10.2±1.8 7 4.14 4.49
Leucine 2.4±0.1 84.3±1.4 69.8±2.3 2.7±0.2 88.8±2.2 204.0±8.0 7.0±0.2 90.3±23 6.7±0.9 63.4±13.3 18 8.38 8.58
Tyrosine 1.9±0.1 76.9±4.3 11.4±0.5 3.1±0.1 67.4±8.1 58.5±1.0 3.9±0.1 62.8±17 3.2±0.5 50.0±11.3 4 5.70 6.11
Phenylalanine 3.1±0.3 76.3±5.3 137.0±4.0 2.4±0.1 82.0±5.1 175.0±17 3.8±0.3 62.6±16 3.7±0.5 47.5±11.4 13 2.61 2.66
Histidine 16.4±0.9 21.0±0.8 62.4±2.7 24.9±0.1 27.8±1.3 71.3±5.4 9.1±0.4 11.9±3.0 2.2±0.4 3.1±0.8 6 3.62 2.98
Hydroxyproline - - - - - - - - - - - - -
Lysine - - - 2.7±0.2 141±8.0 65.4±0.8 20.5±0.8 86.5±24 0.6±0.1 45.9±11 23 8.35 8.72
Arginine 1.3±0.6 196±14 - 1.5±0.2 204±13 120.0±17 9.3±0.3 215±57 3.2±0.4 132±30 49 3.50 3.88
Tryptophan - 18.3±0.5 - 1.4±0.1 19.4±0.5 11.8±1.7 - - - - - - -
Cysteine - - - - - - - - - - - 1.47 1.53
Cystine - - - - - - - - - - 8 - -

PH=Pepsin Hydrolysate; PPH=Pepsin + Pancreatin Hydrolysate; TH=Thermolysin Hydrolysate; *Asx = Asp + Asn; **Glx = Glu + Gln.

58
 Table 2 continued
Cat fish Cat fish Silver carp Visceral Channa striatus muscle Protein Brownstripe red
skin skin (Hypophthalmic protein protein hydrolysates (%) (Ghassem et al., 2011) hydrolysate snapper (Lutjanus
soluble insoluble hthys molitrix) hydrolysates Sarcoplasmic protein hydrolysates myofibrillar protein hydrolysates s from vitta) muscle
hydrolysate hydrolysate by-product of yellowfin produced by produced by Misgurnus protein
Amino acid s (%) s (%) hydrolysate tuna (Thunnus anguilliaca hydrolysates (%)
(Yin et al., (Yin et al., (mg/mL) albacares) udatus (%) (Khantaphant et
2010) 2010) (Duan et al., (g/100 g) (You et al., al., 2011)
2010) (Ovissipour et 2011)
HAP HFP
al., 2010) P T TP P T TP
Aspartic
7.53±0.04 8.87±0.07 2.8077 11.83 8.0±2.12 8.6±1.01 8.1±0.30 9.7±0.44 9.1±0.31 9.4±0.15 8.74 10.65 10.73
acid/Asparagine
Threonine 3.04±0.01 3.91±0.09 1.5110 5.90 4.6±0.10 4.3±0.38 4.5±0.32 4.6±1.10 4.8±0.15 4.7±0.45 4.72 5.10 4.91
Serine 3.11±0.03 3.40±0.16 1.2108 6.81 4.7±2.21 4.5±2.22 4.3±0.41 4.3±0.25 4.3±0.45 4.2±0.24 3.52 5.15 5.02
Glutamic acid/
11.68±0.04 11.80±0.15 5.4271 15.31 8.4±1.92 8.1±1.53 8.1±1.04 16.4±0.30 16.6±0.55 15.6±0.51 16.7 16.23 16.92
Glutamine
Proline 9.49±0.03 5.58±0.39 2.7304 - 3.6±1.55 4.1±2.20 2.8±0.42 3.7±0.26 3.8±0.95 3.5±0.75 5.48 3.66 3.46
Glycine 18.70±0.10 11.94±0.26 5.1012 5.87 8.6±0.10 9.2±1.01 7.6±2.22 4.7±0.55 4.9±0.55 4.7±0.55 5.88 7.48 7.21
Alanine 8.94±0.05 6.56±0.11 2.6944 2.23 7.8±0.10 7.4±2.11 7.2±0.22 6.2±0.11 6.1±2.12 6.0±0.22 7.01 9.38 9.56
Valine 3.67±0.03 5.39±0.05 1.1317 8.93 5.2±0.42 5.2±0.14 7.1±0.32 4.7±2.12 4.8±2.26 4.6±2.01 3.69 5.15 5.15
Methionine 0.44±0.02 1.35±0.02 0.9890 1.48* 2.5±0.21 2.3±0.54 2.1±2.35 3.1±2.75 3.2±2.33 3.1±2.13 2.45 2.93 2.92
Isoleucine 2.61±0.01 4.78±0.03 1.0075 6.93 4.0±1.01 4.4±1.29 7.6±0.45 4.5±0.60 4.5±0.12 4.4±0.65 3.88 4.14 4.07
Leucine 4.42±0.02 7.38±0.03 2.1851 7.70 7.7±0.90 7.7±0.12 7.6±0.22 7.8±0.23 7.9±1.05 7.7±1.22 7.01 8.54 8.62
Tyrosine 1.77±0.00 3.74±0.00 0.8129 1.31 3.8±2.12 2.0±1.00 4.2±1.16 3.3±0.33 3.6±2.12 3.8±1.00 3.5 2.33 2.14
Phenylalanine 2.61±0.41 4.69±0.03 1.4100 3.85 6.1±0.21 6.0±0.42 6.7±0.10 3.4±0.22 3.4±1.01 3.4±1.01 4.14 2.65 2.45
Histidine 1.59±0.00 2.49±0.03 0.5699 8.45 3.7±0.19 3.5±0.43 3.1±0.31 2.1±0.46 2.4±0.23 2.1±0.42 3.90 1.71 1.60
Hydroxylysine 0.70±0.00 0.66±0.20 - - - - - - - - - - -
Lysine 4.62±0.04 6.05±0.11 1.6188 1.87 9.9±0.64 9.5±1.20 7.7±1.33 9.3±0.81 10.4±1.35 10.1±1.34 6.73 9.51 9.76
Arginine 8.81±0.05 8.43±0.12 1.3256 8.81 6.4±0.28 7.3±0.25 7.1±1.45 6.9±0.21 6.6±0.12 6.7±0.12 4.43 5.00 5.06
Tryptophan - - - - 4.2±1.61 2.8±0.20 2.7±0.30 2.8±0.21 3.7±0.43 5.2±0.51 8.23 0.28 0.29
Cysteine - - 0.4290 - 1.0±0.30 0.9±0.31 0.6±0.42 0.3±1.80 0.4±0.20 0.5±0.52 - 0.12 0.14
Cystine - - - - - - - - - - 0.02 - -

HAP=Alcalase/pyloric caeca protease treated hydrolysates; HFP= Flavourzyme/ pyloric caeca protease treated hydrolysates; * = Methionine + cysteine;
P= Proteinase K; T = Thermolysin; TP = Mixture of thermolysin and proteinase K.

59
 Table 2 continued
Mackerel Alaska Protein hydrolysates Tilapia (Oreochromis
(Scomber pollock from Bluewing Searobin niloticus) protein
japonica) frame (Prionotus punctatus) hydrolysates (g/100 g)
defatted protein protein (mg/ 100g protein) (Foh et al., 2011)
Amino acid
hydrolysates hydrolysates (Santos et al., 2011)
(g/100 g) (g/100 g)
(Hou et al., (Hou et al.,
Alcalase Flavourzyme HWDH FMMH
2011a) 2011b)
Aspartic 9.55±0.10 9.65±0.02
9.84 8.38 37.24 ±0.01 29.63 ±0.14
acid/Asparagine
Threonine 4.68 4.05 20.16±0.08 18.50±0.06 4.17±0.04 4.37±0.04
Serine 4.24 5.79 17.45±0.03 16.25±0.02 3.86±0.07 3.87±0.05
Glutamic acid/ 15.65±0.18 17.48±0.04
15.84 14.74 43.25±0.14 33.34±0.25
Glutamine
Proline 3.97 8.32 14.19±0.06 12.44±0.08 4.21±0.03 5.35±0.04
Glycine 4.33 12.04 16.30±0.16 15.38±0.01 4.82±0.03 4.44±0.03
Alanine 8.49 10.47 20.72±0.12 18.95±0.03 6.18±0.05 6.41±0.07
Valine 8.63 6.95 17.82±0.11 16.27±0.08 4.11±0.03 3.96±0.12
Methionine 3.65 3.38 12.22±0.11 10.51±0.07 2.53±0.04 2.87±0.12
Isoleucine 4.25 2.99 18.41±0.17 16.94±0.06 3.22±0.10 3.59±0.06
Leucine 7.49 5.69 30.24±0.11 26.97±0.08 7.81±0.08 7.67±0.08
Tyrosine - - 14.72±0.02 13.73±0.16 2.73±0.06 2.05±0.04
Phenylalanine - - 15.47±0.14 14.75±0.08 3.07±0.05 3.63±0.02
Histidine 2.72 1.44 9.60±0.05 8.54±0.03 2.17±0.06 2.01±0.04
Hydroxylysine - - - - - -
Lysine 7.63 5.41 35.54±0.13 30.14±0.06 9.58±0.03 8.65±0.03
Arginine 5.67 6.47 25.83±0.02 23.13±0.07 5.57±0.06 5.71±0.05
Tryptophan - - 2.54±0.00 3.67±0.02 0.58±0.11 0.28±0.03
Cysteine - - - - 0.61±0.04 0.56±0.05
Cystine - - 2.82±0.00 2.59±0.09 - -
HWDH= Hot water dip hydrolysates; FMMH= Fresh minced meat hydrolysates

60
 Table 3 Antioxidant activities and isolated peptide sequences of various fish protein hydrolysates (FPH) produced from different fish species

Isolated peptide
Part used to Enzymes used to IC50 or % scavenging
Fish species Antioxidant activities showed sequence and Reference
prepare FPH produce FPH values
molecular weight
DPPH radical scavenging activity, linoleic
Scomber
Meat protease N acid autoxidation inhibition activity and --- --- Wu et al. (2003)
austriasicus
reducing power activity.
Mackerel intestine
crude enzyme,
Arg-Pro-Asp-Phe-
Alcalase, α -
Limanda Linoleic acid autoxidation inhibition Asp-Leu-Glu-Pro-Pro-
Frame proteins chymotrypsin, -- Jun et al. (2004)
aspera activity Tyr
papain, pepsin,
Pronase E,
Neutrase and trypsin
Crude proteinase Linoleic acid peroxidation inhibition Hydroxyl radical
Theragra Leu-Pro-His-Ser-Gly-
Frame proteins from mackerel activity scavenging Je et al. (2005)
chalcogramma Tyr (672 Da)
intestine 35 % at 53.6 µM
DPPH radical scavenging activity, Val-Lys-Ala-Gly-Phe-
Alcalase, α -
hydroxyl radical scavenging activity, Ala-Trp-Thr-Ala-Asn-
chymotrypsin,
Tuna Backbones superoxide radical scavenging activity and Gln-Gln-Leu-Ser -- Je et al. (2007)
Neutrase, papain,
lipid peroxidation inhibition activity (1519 Da)
pepsin and trypsin
DPPH radical scavenging activity, Glu-Ser-Thr-Val-Pro- DPPH (IC50=41.37 µM),
Pepsin, trypsin, hydroxyl radical scavenging activity, Glu-Arg-Thr-His-Pro- hydroxyl (IC50=17.77
papain, peroxyl radical scavenging activity, Ala-Cys-Pro-Asp-Phe- µM), peroxyl
Johnius
Frame α-chymotrypsin, superoxide radical scavenging activity and Asn (1801 Da) (IC50=18.99 µM) and Kim et al. (2007)
belengerii
Alcalase and lipid peroxidation inhibition activity superoxide radicals
Neutrase (IC50=172.10 µM).

Alcalase and DPPH radical scavenging activity, Fe2+ Klompong et al.

Selaroides
Body meat Flavourzyme chelating activity, and reducing power -- -- (2007 & 2008)
leptolepis
activity

61
 Isolated peptide
Part used to Enzymes used to IC50 or % scavenging
Fish species Antioxidant activities showed sequence and Reference
prepare FPH produce FPH values
molecular weight
DPPH radical scavenging activity and Souissi et al. (2007)
Sardinella Heads and linoleic acid autoxidation Inhibition -- --
Alcalase
aurita viscera activity

DPPH radical scavenging activity, Fe2+ Thiansilakul et al.

Decapters Ordinary chelating activity, Fe3+ reducing activity -- -- (2007)
Flavourzyme
maruadsi muscle
Linoleic acid autoxidation Inhibition Dong et al. (2008)
Hypophthalmic Alcalase and activity, hydroxyl radical scavenging --- --
Muscle activity, ferrous ions chelating activity
hthys molitrix Flavourzyme

DPPH radical scavenging activity, ABTS Khantaphant and

Proteases from
radical scavenging activity and ferric -- -- Benjakul (2008)
Lutjanus vitta Skin gelatin pyloric caeca of
reducing antioxidant power
crude extract
DPPH radical scavenging activity,
Cryotin F, protease A
Thiobarbituric acid reactive substances
Oreochromis Muscle fillet amano, protease N Raghavan and
(TBARS) preventing activity, Fe2+ -- --
niloticus isolate amano, Flavourzyme Kristinsson (2008)
chelating activity, inhibition of formation
and Neutrase
of lipid hydroperoxides activity
Cryotin F, protease A
ROS scavenging activity, ABTS radical
Oreochromis Muscle fillet amano, protease N Raghavan et al.
scavenging activity and ferric reducing -- --
niloticus isolate amano, Flavourzyme (2008)
antioxidant power (FRAP)
and Neutrase
Papain, pancreatin,
Ctenopharyng Hydroxyl radical scavenging ability and Pro-Ser-Lys-Tyr-Glu- Hydroxyl radical
Muscle bromelain, Neutrase Pro-Phe-Val (966.3 Da)
Ren et al. (2008)
odon idellus lipid peroxidation inhibition activity. (IC50= 2.86 mg/ml)
and Alcalase
Validase, DPPH radical scavenging activity, ABTS
Flavourzyme radical scavenging activity, Oxygen radical
Merluccius Samaranayaka and
Mince and proteolytic absorbing activity, Fe2+ ion chelating -- --
productus Li-chan (2008)
enzymes from Kudoa activity and lipid peroxidation inhibition
paniformis activity.

62
 Isolated peptide
Part used to Enzymes used to IC50 or % scavenging
Fish species Antioxidant activities showed sequence and Reference
prepare FPH produce FPH values
molecular weight

DPPH radical scavenging activity, ferric

reducing antioxidant power, oxygen Theodore,
Ictalurus
Fillets isolate Protamex radical absorbance activity and inhibition -- -- Raghavan, and
punctatus
of formation of thiobarbituric acid reactive Kristinsson (2008)
substances

DPPH radical scavenging activity,

Protamine,
hydroxyl radical scavenging activity and Pro-Arg Hydroxyl radical
Salmon derived from Pancreatin Wang et al. (2008)
superoxide anion radical scavenging (271.3 Da), (21.04 % at 40 µg/mL)
fish milt
activity
Bromelain, papain, DPPH radical scavenging activity, linoleic
Rachycentron acid autoxidation inhibition activity
Skin pancreatin, and -- -- Yang et al. (2008)
canadum
trypsin
Pepsin, alkaline
DPPH radical scavenging activity, Iron
protease, trypsin-like
reducing activity, β-carotene bleaching
protease, crude
Mustelus preventing activity, lipid peroxidation Bougatef et al.
Muscle enzyme extract from -- --
mustelus inhibition activity (2009)
M. mustelus intestine,
bovine trypsin

DPPH radical scavenging activity and β-

Pollachius carotene-linoleate oxidation preventing Chabeaud et al.
Muscle Alcalase -- --
virens activity (2009)

Fe2+ chelating activity, ferric ion

reducing capacity, ABTS radical (2,2- Gimenez et al.
Sole Skin gelatin Alcalase azinobis-(3-ethylbenzothiazoline-6- -- --
(2009)
sulfonic acid)) scavenging capacity

63
 Part used Isolated peptide
Enzymes used to IC50 or % scavenging
Fish species to prepare Antioxidant activities showed sequence and Reference
produce FPH values
FPH molecular weight

Tuna Flavourzyme,
DPPH radical scavenging activity,
(Katsuwonus Liver Alcalase, Protamex -- -- Je et al. (2009)
hydroxyl radical scavenging activity,
pelamis) and Neutrase
hydrogen peroxide scavenging activity and
ferrous ion chelating activity
Pro-Val-Ser-His-Asp-
His-Ala-Pro-Glu-Tyr
DPPH radical scavenging activity and
Cooking (1305 Da), Pro-Ser-
Thunnus tonggol Orientase linoleic acid autoxidation inhibition -- Hsu et al. (2009)
juice Asp-His-Asp-His-Glu
activity
(938 Da), and Val-
His-Asp-Tyr (584 Da)
Selaroides Alcalase and Klompong et al.
Meat In vitro plasmid DNA relaxation activity -- --
leptolepis Flavourzyme (2009a)
Salmo salar,
Onchorhynchus
kisutch, Theragra
Pepsin, pancreatin DPPH radical scavenging activity Nakajima et al.
chalcogramma Muscles -- --
and thermolysin (2009)
and
Micromesistius
australis
DPPH radical scavenging activity and iron
mediated liposomes oxidation reducing
Gadus morhua Backbones Protamex -- -- Slizyte et al. (2009)
activity.

DPPH radical scavenging activity and

Skin
Tilapia Thermal hydrolysis linoleic acid autoxidation inhibition -- -- Yang et al. (2009)
gelatin
activity

DPPH radical scavenging activity,

Misgurnus hydroxyl radical scavenging activity,
Meat Papain and Protamex -- -- You et al. (2009)
anguillicaudatus ABTS radical scavenging activity and
reducing power activity

64
 Part used Isolated peptide
Enzymes used to IC50 or % scavenging
Fish species to prepare Antioxidant activities showed sequence and Reference
produce FPH values
FPH molecular weight

DPPH radical scavenging activity,

Superoxide anion radical scavenging
Theragra Skin Trypsin and
activity, hydroxyl radical scavenging -- -- Zhuang et al. (2009)
chalcogramma collagen flavorzyme
activity and hydrogen peroxide scavenging
activity

DPPH radical scavenging activity,

Alcalase, Neutrase,
Tuna peroxide and hydroxyl radical scavenging
Liver Protamex and -- -- Ahn et al. (2010)
activity and reducing power
Flavourzyme
DPPH radical scavenging activity,
By
Aphanopus carbo Protamex Reducing reducing activity, hydroxyl -- -- Batista et al. (2010)
products
radical scavenging activity
Alcalase, enzyme
DPPH radical scavenging activity
Heads and extracts from
Sardinella aurita Ferric (Fe3+) reducing antioxidant activity, -- -- Barkia et al. (2010)
viscera sardinelle, cuttlefish
β-Carotene Bleaching inhibition activity
and smooth hound
Leu-His-Tyr,
Alcalase, crude DPPH radical-
Leu-Ala-Arg-Leu,
enzyme preparations scavenging activity (63 ±
Heads and DPPH radical scavenging activity, linoleic Gly-Gly-Glu,
from Aspergillus 1.57 % at 150 µg/ml) Bougatef et al.
Sardinella aurita viscera acid autoxidation inhibition activity and Gly-Ala-His,
clavatus, B. among these peptides (2010)
proteins reducing power activity Gly-Ala-Trp-Ala, Pro-
licheniformis, and first peptide showed
His-Tyr-Leu and Gly-
viscera of Sardina highest activity
Ala-Leu-Ala-Ala-His
pilchardus
Leu-Pro-Thr-Ser-Glu-
DPPH radical scavenging activity and DPPH radical
Ala-Ala-Lys-Tyr (978
Dark Orientase and linoleic acid autoxidation inhibition scavenging activity (79.6
Thunnus tonggol Da) Hsu (2010)
muscle protease activity % and 85.2 % at 100
and Pro-Met-Asp-Tyr-
µg/ml)
Met-Val-Thr (756 Da)

65
 Part used Isolated peptide
Enzymes used to IC50 or % scavenging
Fish species to prepare Antioxidant activities showed sequence and Reference
produce FPH values
FPH molecular weight

Asp-Pro-Ala-Leu-Ala- DPPH (IC50 =8.82 µM),

Oreochromis Scale Alcalase, Pronase E, DPPH radical scavenging activity,
Thr-Glu-Pro-Asp-Pro- Hydroxyl (IC50 =7.56 µM), Ngo et al. (2010)
niloticus gelatin trypsin and pepsin hydroxyl radical scavenging activity and
Met-Pro-Phe (1382.57 and superoxide radicals
superoxide radical anion scavenging
Da) (IC50 =17.83 µM)
activity

DPPH radical scavenging activity, ABTS

Alcalase, Neutrase, radical scavenging activity, Ferric reducing
Priacanthus Skin pyloric caeca extract antioxidant power (FRAP) and linoleic Phanturat et al.
-- --
macracanthus gelatin from Priacanthus acid autoxidation inhibition activity and (2010)
macracanthus lecithin liposome oxidation preventing
activity

DPPH radical scavenging activity and

North Atlantic Bacterial beta-carotene-linoleate oxidation
Skin -- -- Picot et al. (2010)
lean fish endopeptidase preventing activity

DPPH radical scavenging activity, lipid

Lepturacanthus
peroxidation inhibition activity, hydroxyl
savala and Muscle Rajaram and
Pepsin radical scavenging activity and reducing -- --
Sphyraena protein Nazeer (2010)
power activity
barracuda

DPPH radical scavenging activity,

hydroxyl radical scavenging activity,
Papain, pepsin and
Misgurnus Meat ABTS radical scavenging activity, -- -- You et al. (2010a)
pancreatin
anguillicaudatus reducing power activity and Cu2+ ion
chelating activity

DPPH radical scavenging activity,

Hydroxyl radical
Misgurnus hydroxyl radical scavenging activity, Cu2+ Pro-Ser-Tyr-Val
Meat Papain (IC50=1.86 mg/ml) You et al. (2010b)
anguillicaudatus ion chelating activity and linoleic acid (464.2 Da)
autoxidation inhibition activity

66
 Part used Isolated peptide
Enzymes used to IC50 or % scavenging
Fish species to prepare Antioxidant activities showed sequence and Reference
produce FPH values
FPH molecular weight

Theragra Alcalase, Neutrase, DPPH radical scavenging activity and

chalcogramma Skin trypsin, papain, Ferric (Fe3+) reducing antioxidant power -- -- Jia et al. (2010)
pepsin, Flavourzyme,
and Protamex
DPPH radical scavenging activity,
hydroxyl radical scavenging activity,
Misgurnus
Meat Papain Cupric ion chelating activity and linoleic -- -- You et al. (2011)
anguillicaudatus
acid oxidation inhibition activity

Tuna and Halibut Skin Alcalase, Ferric reducing antioxidant power (FRAP), Aleman et al.
gelatin collagenase, trypsin and ABTS radical scavenging activity -- -- (2011)
and pepsin
Silver carp Processing Alcalase, DPPH radical scavenging activity,
(Hypophthalmicht by- Flavourzyme, hydroxyl radical scavenging activity, Zhong et al.
hys molitrix) products Neutrase, Protamex, Superoxide anion radical scavenging -- -- (2011)
papain, pepsin and activity and linoleic acid autoxidation
trypsin inhibition activity

Lutjanus vitta Muscle Alcalase, DPPH radical scavenging activity,

Flavourzyme and ABTS radical scavenging activity, Khantaphant et al.
pyloric caeca Ferric reducing antioxidant power (FRAP), -- -- (2011)
protease Ferrous ion chelating activity,
β-Carotene linoleic acid emulsion model
system and Lecithin liposome model

Nemipterus Muscle Skipjack tuna pepsin DPPH radical scavenging activity, Nalinanon et al.
hexodon ABTS radical scavenging activity and -- -- (2011)
Ferrous ion chelating activity

Parastromateus Viscera Pepsin, trypsin, and DPPH radical scavenging activity, Ala-Met-Thr-Gly-Leu- DPPH radical Jai Ganesh et al.
niger α-chymotrpsin Fe2+ chelating activity and Ferric (Fe3+) Glu-Ala (701.9 Da) Scavenging activity (2011)
reducing antioxidant power (78.6 %)

67
 Part used Isolated peptide
Enzymes used to IC50 or % scavenging
Fish species to prepare Antioxidant activities showed sequence and Reference
produce FPH values
FPH molecular weight
Magalaspis Skin Pepsin, trypsin and α- Ferric (Fe3+) reducing antioxidant activity Asn-His-Arg-Tyr- Sampath Kumar
cordyla and chymotrpsin and Fe2+ chelating activity Asp-Arg (856 Da), et al. (2011)
Otolithes ruber Gly-Asn-Arg-Gly- --
Phe-Ala-Cys-Arg-His-
Ala (1101.5 Da)
Sphyraena Backbones Papain, pepsin and DPPH radical scavenging activity, Ferric Nazeer et al.
barracuda and trypsin (Fe3+) reducing antioxidant power and -- -- (2011)
Lepturacanthus Lipid peroxidation inhibition activity
savala
Nemipterus Muscle Papain, pepsin and DPPH radical scavenging activity, Ferric Naqash and
japonicus trypsin (Fe3+) reducing antioxidant power, Fe2+ Nazeer (2011a)
chelating activity and Lipid peroxidation -- --
inhibition activity

Exocoetus Backbone Papain, pepsin and DPPH radical scavenging activity, Lipid Naqash and
volitans trypsin peroxidation inhibition activity Nazeer (2011b)
Superoxide anion radical scavenging -- --
activity and hydroxyl radical scavenging
activity

Catla catla and Visceral Protease-P-amano 6, DPPH radical scavenging activity and total -- -- Hathwar et al.
Labeo rohita waste Alcalase, protex 7L, antioxidant activity (2011)
and Neutrase

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 Table 4 Brand names, particulars and health promoting applications of commercially available
nuraceuticals/health foods/functional foods in different countries produced from fish protein hydrolysates

Product brand Particulars Nutraceutical applications Country Reference

name

Seacure®, Prepared by hydrolyzing Dietary supplement helps to support the US and Marchbank et al.
deep ocean white fish cells in the gastrointestinal tract and Canada (2008)
proteins regulate bowel functions.

Amizate® Produced from Atlantic Sports nutrition (supports the body’s North Nesse et al. (2011)
salmon fish proteins by muscle anabolism and metabolic America
autolysis recovery).

Stabilium® 200 Prepared from Molva Supports the body’s response to stress and UK Guerard et al.
dypterygia by autolysis provides nutritional support for memory (2010)
and cognitive function.

PROTIZEN® Produced by enzymatic It is "mood food" and dietary supplement UK Guerard et al.
hydrolysis of white fish to fight against stress and its symptoms (2010)
proteins (weight disorders, work pressure, sleep
troubles, concentration difficulties and
mood troubles).

Produced from Bonito It supports healthy vascular function for US and www.metagenics.co
Vasotensin® (Sarda orientalis) by optimal blood flow and healthy blood Japan m
thermolysin hydrolysis pressure levels.

Produced from Bonito It lowers the blood pressure by inhibiting US and www.naturalfactors.
PEPTACE® (Sarda orientalis) by ACE enzyme. Japan com
thermolysin hydrolysis

Manufactured by It helps in the blood glucose stabilization, UK and Guerard et al.

Nutripeptin® enzymatic hydrolysis of and weight management. USA (2010)
Cod fish fillet/muscle
protein

LIQUAMEN® Prepared from Molva Dietary supplement that helps in reducing UK Guerard et al.
molva by autolysis oxidative stress, lowering glycemic index (2010)
and anti-stress.

Produced from North Dietary supplement recommended for UK www.dielen.fr

MOLVAL® Atlantic fish Molva cholesterol equilibrium, stress control and
molva by enzymatic promotes good cardiovascular health.
hydrolysis

SEAGEST® Prepared by hydrolyzing It supports the structure of the intestinal US www.trimedica.com

deep ocean white fish lining and promotes intestinal health.
proteins

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